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Methods in
Molecular Biology 2294
Metastasis
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK
Edited by
Ulrike S. Stein
Translational Oncology of Solid Tumors Experimental and Clinical Research Center,
Charité – Universit€atsmedizin Berlin and Max-Delbrück-Center for Molecular Medicine, Berlin, Germany
Editor
Ulrike S. Stein
Translational Oncology of Solid
Tumors Experimental and Clinical
Research Center
Charité – Universit€atsmedizin Berlin
and Max-Delbrück-Center for Molecular Medicine
Berlin, Germany
This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer
Nature.
The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.
Preface
Cancer metastasis is the most lethal attribute of cancer. It is estimated that at least 90% of all
cancer patients dying from any type of cancer are dying due to cancer metastasis. Cancer
metastasis is both the process and the outcome of the process, whereby cancer cells leave a
tumor at the primary or original site, spread throughout the body, and seed tumors in new
organs or secondary sites. It is mainly metastasis that makes cancer lethal. Although there is no
cure for metastasis, only 5% of cancer research funding worldwide is devoted to metastasis
research.
With this book edition we will enable current and future basic and translational
researchers, clinical scientists, and oncologists to take the challenges and work in this field
of research. This book series is devoted to disseminate Methods and Protocols, with focus
on those indispensable for state-of-the-art metastasis research.
The first part introduces models useful for metastasis research, starting with Zebrafish
models for cancer cell invasion and metastasis, the chicken egg chorioallantoic membrane
(CAM) model for investigating invasion and the metastasis cascade, in vitro 3D models for
tunable stiffness, patient-derived mouse xenografted (PDX) metastasis models from solid
tumors for predicting therapy response, and organotypic hippocampal slices to analyze brain
metastasis and primary brain tumor growth. In the second part, functional in vitro assays are
described, illustrating the steps of the metastatic cascade, such as cancer cell mechanical
properties, matrix degradation, cancer cell adhesion, migration, and invasion, platelet
aggregation, blood-based pro-metastasis immune cells, bone marrow-derived macrophages,
and cancer cell extravasation. The third part describes options for prognostication of
metastasis, e.g., by using bioinformatics pipelines to identify metastatic biomarkers, barcod-
ing technology and in vivo assessment for analyzing metastatic abilities and metastatic cell
potential, as well as nucleolar contents and proteases to uncover metastatic progression. A
last, fourth part complements the book outline with clinical methods, such as imaging
procedures and the path toward metastasis research from the clinical setting showing a
clinicians perspective.
Finally, we would very much appreciate if also the cancer patient her/himself—who
certainly is the center of all efforts—might benefit from this book. Since when the patient is
asking “Has it spread?,” she/he is actually asking: “What is my prognosis? Do I have therapy
options? Will I survive?” The hope is that this book edition will contribute to a more often
optimistic answer to the patient.
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
vii
viii Contents
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
Contributors
ix
x Contributors
Metastasis Models
Chapter 1
Abstract
Cancer cell vascular invasion and extravasation at metastatic sites are hallmarks of malignant progression of
cancer and associated with poor disease outcome. Here we describe an in vivo approach to study the invasive
ability of cancer cells into the vasculature and their hematogenous metastatic seeding in zebrafish (Danio
rerio). In one approach, extravasation of fluorescently labeled cancer cells is monitored in zebrafish embryos
whose vasculature is marked by a contrasting fluorescent reporter. After injection into the precardiac sinus
of 2-day-old embryos, cancer cells can extravasate from the vasculature into tissues over the next few days.
Extravasated cancer cells are identified and counted in live embryos via fluorescence microscopy. In a second
approach, intravasation of cancer cells can be evaluated by changing their injection site to the yolk sac of
zebrafish embryos. In addition to monitoring the impact of drivers of malignant progression, candidate
inhibitors can be studied in this in vivo model system for their efficacy as well as their toxicity for the host.
Key words Cancer, Extravasation, Zebrafish, Embryo, Fluorescence, Vascular Invasion, Metastasis
1 Introduction
Ulrike S. Stein (ed.), Metastasis: Methods and Protocols, Methods in Molecular Biology, vol. 2294,
https://doi.org/10.1007/978-1-0716-1350-4_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021
3
4 Sarah Martinez Roth et al.
overlaps with the human genome by 70%, and most organs and
cellular function are conserved. An overview of zebrafish in bio-
medical research is provided in an award-winning short movie for
an educated lay audience [29] (www.zebrafishfilm.org). One major
benefit of the model is the potential for cost efficient, relatively high
throughput using dozens of easy-to-visualize transparent embryos
that can grow and develop in small volumes accommodated by
96-well format dishes in a single experiment [30]. The model also
allows the observation of very few to single-cell behaviors that help
elucidate the impact of subpopulations present in heterogeneous
tumor cell populations (see, e.g., Fig. 3) [32]. Therefore, trans-
plantation of cells in the optically clear, immune-permissive zebra-
fish embryos can provide a unique perspective to understand cancer
metastasis and to visualize single-cell cancer progression in real time
[33]. The zebrafish model of cancer invasion and metastasis can
also be used to assess drug effects or screen for candidate inhibitors
in an in vivo model. Drugs to be analyzed can be added to the
growth media of the fish embryos at an appropriate time to read out
the effect on the host embryo as well as the cancer cells transplanted
for evaluation of invasion and metastasis. Several examples from
different areas of study were published during the past years [34–
37] including the application to individualized analysis of human
cancer biopsies [38].
In the approach described, first we utilize a transparent zebra-
fish strain that has its endothelia tagged with a green reef coral
fluorescent protein reporter driven by the kdrl promoter, the recep-
tor for vascular endothelial growth factor [39]. The vascular inva-
sive ability of a commonly used cancer cell line is shown as a
representative example, and the general steps are outlined in
Fig. 1. In this example, MDA-MB-231, a human breast cancer
cell line, was labeled with a red fluorescent lipophilic dye and
injected into the precardiac sinus of 2-day-old embryos. Between
48 and 96 h after the injection, cancer cells that have extravasated
out of the vasculature and invaded into tissues in the caudal region
of embryos can be scored efficiently on a fluorescent microscope
(Fig. 2). In the experiment shown here, the breast cancer cell line
was cultured at different cell densities for a few days resulting in
inhibition of the Hippo pathway and in vitro invasion of an endo-
thelial monolayer by cells grown at low density [31]. In the zebra-
fish, embryos cells grown at low density extravasated from the
vasculature and invaded tissues at a significantly higher rate con-
firming the biologic significance of the in vitro findings (Fig. 2b, c).
In a second approach, in wild-type zebrafish embryos without
fluorescent vasculature, the injection site of cancer cells is changed
to the yolk sac (Fig. 3a, b). The approach allows for the study of
heterogeneous cancer cell populations that are labeled with differ-
ently colored Q-dots (Fig. 3). Cancer cells that have intravasated in
6 Sarah Martinez Roth et al.
Fig. 1 Overview of the cancer cell extravasation assay in zebrafish embryos. Cancer cells are incubated with
Q-dots (Thermo Fisher) or a fluorescent dye that is taken up by the cells as a label (red). Cells in suspension
are injected into the precardiac sinus of the embryos (2 days old). During the next 2–4 days, cancer cells traffic
in the vasculature can invade tissues in the caudal region of the embryo and are imaged after mounting in an
anesthetic agarose medium. The vascular endothelia in the embryos express GFP (Green Fluorescent Protein)
to identify the location of cancer cells either inside the vasculature or inside the tissues by confocal
microscopy. (Reproduced with permission; Ref. [40])
the yolk sac can traffic in the vasculature and are scored in the caudal
region (Fig. 3c) 24–48 h after injection [31, 41]. In the example
shown here, breast cancer cells grown at low or high cell density
were labeled with green or red Q-dots, respectively and
Zebrafish Models to Study Drivers of Metastasis 7
Fig. 3 Intravasation of two distinct cancer cell populations after yolk sac injection. (a) Brightfield image of a
wild-type zebrafish embryo 4 days after fertilization. The boxed areas indicate the yolk sac. (b) Yolk sac with
green (invasive ¼ low-density growth) and red (noninvasive ¼ high-density growth) Q-dot labeled cancer
cells. (c) The caudal region with cancer cells that intravasated into the vasculature and reached the tail region.
(d) The percentage of embryos (n ¼ 75) with intravasated cells in the caudal region 2 days after injection
(Original data in Ref. [31])
co-inoculated at the same time into the yolk sacs of embryos for a
direct comparison (Fig. 3b). Cells grown at low density intravasated
at a significantly higher rate than the high-density cells as evidenced
by their appearance in the tail vasculature (Fig. 3c, d). The extrava-
sation and intravasation assays from Figs. 2 and 3 provide comple-
mentary insights. Both approaches can also be used to assess the
contribution of pathways by knockdown of candidate genes as well
as treatment with small molecule or antibody-based inhibitors of
potential driver molecules. Examples of such interventions can be
found in [31, 41].
8 Sarah Martinez Roth et al.
2 Materials
2.1 Injection Station 1. Needles: Capillary Glass, Standard, 1.2 MM 0.68 MM, 400 ,
A-M Systems, Inc., pulled using David Kopf 700C Vertical
Pipette Puller, Hofstra Group. Pull long-tapered pipettes
using 20 mAmp current and a two-coil heating element.
2. Machinery needed for injection station setup: Electrode stor-
age jar, 1.0 MM, World Precision Instruments, Inc., latex
rubber bulbs, 2 mL, Pack of 72, Heathrow Scientific and
micromanipulator, Narishige, Picospritzer II, General Valve
Corporation.
2.2 Manipulating 1. Tools for manipulating embryos: Eyelash Brush, Ted Pella,
Fish Inc., transfer pipettes, and 5 3/400 disposable Pasteur pipettes,
borosilicate glass.
2. Fish water: 0.3 g/L, Instant Ocean Salt, Sea Salt, Pentair. Store
at room temperature for up to 3 months.
3. Tricaine stock solution: Ethyl 3-aminobenzoate methanesulfo-
nate salt (Tricaine, MS-222), Fluka: (4 mg/mL,10 mM Tris,
pH 7) to 50 mL of fish water plus penicillin-G potassium and
streptomycin sulfate.
2.3 Visualization 1. Microscopy method used: For visualizing cells, use Leica SP8
Confocal Microscope.
2. Staining cells:
(a) For staining cancer cells for pre-cardiac sinus injection,
cancer cells were labeled with a lipophilic red fluorescent
dye: Vybrant DiI Cell-Labeling solution (Thermo Fisher).
(b) For staining cancer cells for yolk sac injection, cells were
labeled with 565 nm or 655 nm Q-dots using the
Qtracker Cell-Labeling kit (Thermo Fisher).
3 Methods
3.1 Set up Embryos 1. Separate males and females the night before experiment in a
for Injection and Make breeding cage. The goal is to develop enough zebrafish larvae
Stock Solutions to assess cancer cell vascular invasion.
2. Pull the gate on the breeding cage of pairwise or group in-cross
mating with Tg(kdrl:grcfp)zn1;mitfab692;ednrb1b140 fish (see
Note 1).
Zebrafish Models to Study Drivers of Metastasis 9
3.2 Labeling Cells 1. Grow cells of interest in their recommended culture conditions
with Red Lipophilic (see Note 6).
Fluorescent Dye 2. Generate a single-cell suspension by dissociating an adherent
culture of cancer cells.
3. Wash cells with 1 PBS and add 0.05% trypsin-EDTA solution
(see Note 7).
10 Sarah Martinez Roth et al.
3.5 Mounting 1. Melt 1.5% agarose/2 tricaine solution, and bring to 37 ˚C.
Zebrafish onto Slides 2. Anesthetize the embryo by placing it in 2 tricaine solution.
and Subsequent
3. Transfer the embryo in a drop of 2 tricaine solution to the
Fluorescence Imaging
imaging station (see Note 15).
4. Use a glass pipette to remove the excess 2 tricaine solution,
retaining the embryo on the imaging surface.
5. Overlay one drop of melted agarose solution over the embryo.
6. Before the agarose polymerizes, use an eyelash brush, to orient
the embryo laterally for imaging, making sure the embryo is
flattened along the imaging surface.
7. Submerge the now polymerized agarose drop under 2 tricaine
solution.
8. Image the zebrafish embryo using microscopy.
3.6 Alternative 1. Prepare the microinjection dispense system and injection plates
Application: Injecting as previously described in Subheading 3.3 of this protocol.
Cancer Cells into the 2. Label two cell populations with contrasting fluorescent Q-dots
Yolk Sac from the Qtracker Cell-Labeling kit (Thermo Fisher) or with
fluorescent dyes as described in Subheading 3.2 above.
3. Inject 5–10 nL of 2 107 cells/mL into the yolk sac (Fig. 3b).
Keep the injection volume constant to inject identical cell
numbers (100–200 cancer cells) from each cell population
(see Note 16).
4. Collect the injected embryos as previously described in Sub-
heading 3.3 of this protocol and then screen for successful
injections.
5. Screen and transfer viable embryos that were successfully
injected to a new dish (see Note 17).
6. Transfer the viable embryos to a new petri dish if cancer cells
are clearly seen in the yolk sac (Fig. 3b).
Zebrafish Models to Study Drivers of Metastasis 13
4 Notes
Acknowledgments
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tal regulation of tumor progression and metas- tonakis IK, Sudo R, Kamm RD, Chung S
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5. Hooper S, Marshall JF, Sahai E (2006) Tumor ture of multiple cell types on surfaces or within
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Henke RT, Hohn S, Toretsky JA, Riegel AT, Cancer 7:659–672
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11. Mohanty S, Xu L (2010) Experimental metas- and clinical functional imaging data. elife 7:
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Hayes MN, Garcia EG, Yordán NT,
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21. Dondossola E, Alexander S, Holzapfel BM, Kont YS, Minas TZ, Swift M, Paige M,
Filippini S, Starbuck MW, Hoffman RM, Glasgow E, Toretsky JA, Bosch J (2015) Iden-
Navone N, De-Juan-Pardo EM, Logothetis tification of novel ezrin inhibitors targeting
CJ, Hutmacher DW, Friedl P (2018) Intravital metastatic osteosarcoma by screening open
microscopy of osteolytic progression and ther- access malaria box. Mol Cancer Ther
apy response of cancer lesions in the bone. Sci 14:2497–2507
Transl Med 10:eaao5726
35. Chen C, Choudhury S, Wangsa D, Lescott CJ,
22. Warren SC, Nobis M, Magenau A, Mohammed Wilkins DJ, Sripadhan P, Liu X, Wangsa D,
YH, Herrmann D, Moran I, Vennin C, Con- Ried T, Moskaluk C, Wick MJ (2017) A multi-
way JR, Mélénec P, Cox TR, Wang Y (2018) plex preclinical model for adenoid cystic carci-
Removing physiological motion from intravital noma of the salivary gland identifies
16 Sarah Martinez Roth et al.
Abstract
The CAM model enables an in vivo analysis of the individual sub-steps of the metastatic cascade like local
invasion, intravasation, or the establishment of metastasis in particular organs. Incubated fertilized chicken
eggs are inoculated with human tumor cells and further processed for up to 9–10 days. The invasion and
metastasis of these cells is then detected quantitatively with high specificity and sensitivity by means of a
PCR for human ALU sequences, using the genomic DNA isolated from distant portions of the CAM, as
well as from diverse internal organs of the developing embryo.
Key words Chicken chorioallantoic membrane, ALU elements, DNA, Quantification, TaqMan,
Primer, Probe, Real-time PCR, Metastasis, In vivo model
1 Introduction
Ulrike S. Stein (ed.), Metastasis: Methods and Protocols, Methods in Molecular Biology, vol. 2294,
https://doi.org/10.1007/978-1-0716-1350-4_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021
17
18 Jörg H. Leupold et al.
2 Materials
2.1 Equipment 1. Digital egg incubator for chicken, fully automatic turn system.
2. Scriber with pen-like handle and diamond tip.
3. Pencil.
4. Egg piercer tool.
5. Mini rotary tool with cutting wheel.
6. Set of nickel-plated steel cover glass tweezers, curved.
7. Dark room.
8. Egg candling lamp with LED light.
9. Workstation with white lamp and UV sterilization.
10. Set of nickel-plated steel microscopy scissors, pointed, straight.
11. Set of nickel-plated steel tweezers, curved.
12. Digital shaking thermo block.
13. Digital shaking water bath.
14. Laboratory rotor homogenizer with disposable, plastic-based
generators.
15. Spectrophotometer.
16. Real-time thermal cycler.
17. Sterile work bench.
18. Vortexer.
19. Standard laboratory centrifuge or microfuge.
20 Jörg H. Leupold et al.
3 Experimental Procedure
For each experiment, include a separate set of eggs that can be used
for the isolation of genomic chicken DNA for standard curve
preparation. For statistically valid results, a minimum of 10 eggs/
group is recommended, and the experiment must be carried out as
three independent biological replicates.
In Vivo Investigation of Metastasized Cells 21
3.3 Isolation Preheat water bath to 55 C for use in step 4 and 37 C for use in
of Genomic DNA step 6. Prechill a centrifuge suitable for 13 ml round-bottom tubes
and equipped with a swinging rotor to 4 C for use in step 9.
Preheat shaking thermo block to 65 C for use in step 19.
1. Resolving cleaning solution.
2. Homogenize each tissue individually with a laboratory rotor
homogenizer (see Note 9). Avoid extensive foam formation. If
disposable, plastic-based generators are used, they should be
immediately placed into “cleaning solution” to avoid later
impurity from dried albumin and proteins.
3. Immediately add 20 μl proteinase K to the homogenate and
invert the tube 25 times to mix the solution equally.
4. Place the tubes in a water bath with orbital shaking motion and
incubate the samples overnight at 55 C until the tissue has
completely lysed.
5. Add 20 μl RNase A solution, and mix the samples by inverting
25 times.
6. Further incubate the samples at 37 C for 1 h in a water bath
with orbital shaking motion.
7. Cool down the samples on crushed ice up to 3 min.
8. Add 1.25 ml of protein precipitation solution (provided by
Gentra Purgene Tissue Kit) to each sample and vortex vigor-
ously for 20 s.
9. Centrifuge the samples in a swinging rotor at 4 C for 15 min at
2000 g. If the precipitated proteins do not form a tight
pellet, incubate the tubes for additional 5 min on crushed ice
and repeat centrifugation.
10. Transfer the supernatants to 15 ml centrifuge tubes without
disturbing the pellet and add 4 ml of isopropanol to the
supernatant.
11. Mix gently by inverting the tubes not less than 50 times or
until a white, cloudy precipitate becomes visible.
In Vivo Investigation of Metastasized Cells 23
3.4 TaqMan®-Based Make sure that your quantitative PCR machine is able to detect the
Quantitative PCR fluorescent reporter dye FAM and the fluorescent quencher dye
of Samples TAMRA. Dilute primers and probe to a working stock concentra-
and Standard Curve tion of 10 μM. Hold available genomic DNA from untreated
chicken eggs and each human cell line (concentration equal to
2 106 cells/100 μl) used in your current experimental setup. It
is generally recommended to use at least three technical replicates
for each PCR.
1. Dilute the genomic chicken DNA of all test samples to a final
concentration of 1 μg/5 μl in water.
2. Prepare a serial dilution using genomic DNA from the respec-
tive cell line (see Note 10). For this six 1.5 ml tubes are primed
with 45 μl water containing 9 μg genomic chicken DNA
isolated from untreated eggs. An additional 1.5 ml tube is
pipetted in a way that it contains genomic human cell DNA
24 Jörg H. Leupold et al.
4 Notes
1. Prevent foam while making SDS containing buffer and use SDS
pellets instead of powder to avoid inhaling of SDS dust leading
to possible respiratory tract irritation.
2. Before even looking at incubator brands, it should be deter-
mined how many eggs can be incubated at once. Incubators
come in many different sizes and shapes, starting with only a
few eggs and running all the way up to a few thousand.
In Vivo Investigation of Metastasized Cells 25
Acknowledgments
H.A. was supported by the Alfried Krupp von Bohlen und Halbach
Foundation, Essen, the Deutsche Krebshilfe, Bonn (70112168),
the Deutsche Forschungsgemeinschaft (DFG, grant number AL
465/9-1), the HEiKA Initiative (Karlsruhe Institute of Technol-
ogy/University of Heidelberg collaborative effort), Dr. Hella-Büh-
ler-Foundation, the DKFZ-MOST Cooperation, Heidelberg
(grant number CA149), the HIPO/POP-Initiative for Persona-
lized Oncology, Heidelberg (H032 and H027).
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Giovannetti E (2017) Development of
Chapter 3
Abstract
Three-dimensional models of spheroid formation have been routinely used in the cancer field to test the
colony forming capacity of malignant cells in an in vitro setting. Use of such a model provides a robust
surrogate for in vivo testing, enabling large-scale interrogation into the effect of certain treatment condi-
tions. This adapted protocol describes a high throughput and readily accessible composite alginate hydrogel
system for spheroid formation, within a biomechanically tunable three-dimensional environment. This
model therefore allows users to examine the effect of certain treatment conditions while cells are embedded
within a hydrogel of defined stiffness. This is particularly important in the context of cancer where cells
experience a wide range of mechanical properties within their microenvironment, driven by widespread
changes in the extracellular matrix composition and architecture.
This protocol describes a high-throughput method which results in homogeneous interpenetrating
polymer networks of collagen and alginate. We show that this network readily supports single-cell spheroid
formation in numerous malignant cell lines (breast cancer, lung cancer, and melanoma) and that these can
be robustly analyzed for colony formation measures such as spheroid size, spheroid number, and overall cell
viability; therefore, allowing users to undertake high-throughput, in vitro screening against a controlled
biomechanical background.
Key words Alginate, Spheroid, Colony formation, Stiffness, Extracellular matrix, High throughput
1 Introduction
Ulrike S. Stein (ed.), Metastasis: Methods and Protocols, Methods in Molecular Biology, vol. 2294,
https://doi.org/10.1007/978-1-0716-1350-4_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021
27
28 Elysse C. Filipe et al.
2 Materials
3 Method
3.1 Reagent 1. Prepare a 2% alginate stock solution in saline and leave under
Preparation gentle agitation overnight at room temperature. Once dis-
solved, solution should be filter-sterilized (0.45 μm filter) in a
sterile working environment and stored at 4 C for future use.
We have successfully used alginate stock solutions prepared
3 weeks prior to an experiment and stored (maintaining steril-
ity) at 4 C until use (see Note 1).
2. Prepare a 1 M CaCO3 stock solution in water and autoclave. In
a sterile working environment, aliquot into smaller working
stocks for routine use.
Note: This should be prepared in a 1 L glass bottle to avoid
over boiling during the autoclaving cycle.
3. Prepare a collagen working solution at 2 mg/mL collagen I in
17.4 mM acetic acid.
Note: Ensure you work continuously on ice to avoid pre-
cipitation of the collagen.
4. Prepare a 220 mM NaOH stock solution in water. This solu-
tion should be filter-sterilized (0.22 μm) in a sterile working
environment prior to use.
34 Elysse C. Filipe et al.
Table 1
Volumes to prepare 2, 5, or 10 mLs of alginate plugs
One calf’s foot, a pound and a half or two pounds of neck of veal
or beef, a small onion, a carrot, a bunch of parsley, a little spice, a bit
or two of quite lean ham, dressed or undressed, and five half pints of
water, boiled very slowly for five or six hours will give a strong,
though not a highly-flavoured jelly. More ham, any bones of unboiled
meat, poultry, or game will, in this respect, improve it; and the liquor
in which fowls or veal have been boiled for table should, when at
hand, be used for it instead of water. These jellies keep much better
and longer when no vegetables are stewed down in them.
GLAZE.
Sauces.
INTRODUCTORY REMARKS.
When this is done with the yolks of eggs, they should first be well
beaten, and then mixed with a spoonful of cold stock should it be at
hand, and with one or two of the boiling sauce, which should be
stirred very quickly to them, and they must in turn be stirred briskly to
the sauce, which may be held over the fire, and well shaken for an
instant afterwards, but never placed upon it, nor allowed to boil.
To the roux or French thickening (which follows), the gravy or other
liquid which is to be mixed with it should be poured boiling and in
small quantities, the saucepan being often well shaken round, and
the sauce made to boil up after each portion is added. If this
precaution be observed, the butter will never float upon the surface,
but the whole will be well and smoothly blended: it will otherwise be
difficult to clear the sauce from it perfectly.
For invalids, or persons who object to butter in their soups or
sauces, flour only mixed to a smooth batter, and stirred into the
boiling liquid may be substituted for other thickening: arrow-root also
used in the same way, will answer even better than flour.
FRENCH THICKENING, OR BROWN ROUX.
Sauce tournée is nothing more than rich pale gravy made with
veal or poultry (see Consommé, Chapter IV.) and thickened with
delicate white roux. The French give it a flavouring of mushrooms
and green onions, by boiling some of each in it for about half an hour
before the sauce is served: it must then be strained, previously to
being dished. Either first dissolve an ounce of butter, and then
dredge gradually to it three-quarters of an ounce of flour, and
proceed as for the preceding receipt; or blend the flour and butter
perfectly with a knife before they are thrown into the stewpan, and
keep them stirred without ceasing over a clear and gentle fire until
they have simmered for some minutes, then place the stewpan high
over the fire, and shake it constantly until the roux has lost the raw
taste of the flour; next, stir very gradually to it a pint of the gravy,
which should be boiling. Set it by the side of the stove for a few
minutes, skim it thoroughly, and serve it without delay.
Butter, 1 oz.; flour, 3/4 oz.; strong pale gravy, seasoned with
mushrooms and green onions, 1 pint.
Obs. 3.—With the addition of three or four yolks of very fresh
eggs, mixed with a seasoning of mace, cayenne, and lemon-juice,
this becomes German sauce, now much used for fricassees, and
other dishes; and minced parsley (boiled) and chili vinegar, each in
sufficient quantity to flavour it agreeably, convert it into a good fish
sauce.
BÉCHAMEL.
This is a fine French white sauce, now very much served at good
English tables. It may be made in various ways, and more or less
expensively; but it should always be thick, smooth, and rich, though
delicate in flavour. The most ready mode of preparing it is to take an
equal portion of very strong, pale veal gravy, and of good cream (a
pint of each for example), and then, by rapid boiling over a very clear
fire, to reduce the gravy nearly half; next, to mix with part of the
cream a tablespoonful of fine dry flour, to pour it to the remainder,
when it boils, and to keep the whole stirred for five minutes or more
over a slow fire, for if placed upon a fierce one it would be liable to
burn; then to add the gravy, to stir and mix the sauce perfectly, and
to simmer it for a few minutes longer. All the flavour should be given
by the gravy, in which French cooks boil a handful of mushrooms, a
few green onions, and some branches of parsley before it is
reduced: but a good béchamel may be made without them, with a
strong consommé (see pale veal gravy, page 98) well reduced.
Strong pale veal gravy (flavoured with mushrooms or not), 1 pint:
reduced half. Rich cream, 1 pint; flour, 1 tablespoonful: 5 minutes.
With gravy, 4 or 5 minutes.
Obs.—Velouté, which is a rather thinner sauce or gravy, is made
by simply well reducing the cream and stock separately, and then
mixing them together without any thickening.
BÉCHAMEL MAIGRE.
Cut half a pound of veal, and a slice of lean ham or smoked beef,
into small dice, and stew them in butter, with vegetables, as directed
in the foregoing receipt: stir in the same proportion of flour, then add
the milk, and let the sauce boil very gently for an hour. It should not
be allowed to thicken too much before it is strained.
Obs.—Common béchamel, with the addition of a spoonful of
made-mustard, is an excellent sauce for boiled mutton.
RICH MELTED BUTTER.
Pour half a pint of good but not very thick, boiling melted butter to
the well-beaten yolks of two or three fresh eggs, and stir them briskly
as it is added; put the sauce again into the saucepan, and shake it
high over the fire for an instant, but do not allow it to boil or it will
curdle. Add a little lemon-juice or vinegar, and serve it immediately.
NORFOLK SAUCE, OR RICH MELTED BUTTER WITHOUT
FLOUR.
Thicken half a pint of new milk with rather less flour than is
directed for the common melted butter, or with a little arrow-root, and
stir into it by degrees after it has boiled, a couple of ounces of fresh
butter cut small; do not cease to stir the sauce until this is entirely
dissolved, or it may become oiled, and float upon the top Thin
cream, substituted for the milk, and flavoured with a few strips of
lemon-rind cut extremely thin, some salt, and a small quantity of
pounded mace, if mixed with rather less flour, and the same
proportion of butter, will make an excellent sauce to serve with fowls
or other dishes, when no gravy is at hand to make white sauce in the
usual way.
BURNT OR BROWNED BUTTER.