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ZIRC
This material is from the 4th edition of The Zebrafish Book. The 5th edition is available in print and within the ZFIN Protocol Wiki.

CHAPTER 10 - RECIPES


ABC Solution (Avidin-Biotin HRP Complex)
Agar/Sucrose
Atabrine Stock Solution
Blocking Solution for Western Blots
BSA/PBS/DMSO
BT Fix
Chorion Removal
DAB (Diaminobenzidine)
DAB Heavy Metal Stain
DAB Presoak Solution
DAB Solution
DNA Extraction Buffer
EDTA
Egg Water
Embryo Extract
Embryo Medium
Epon
Epon/Araldite
Finquel (MS222)
Fish Water
Fix Buffer
2X Fix Buffer
Fixatives
Gelatin Embedding Medium
Genomic DNA Extraction Buffer
GHCl Buffer
Giemsa
Ginzburg Fish Ringers
GIT Buffer
Growth Medium
Hank's (Final)
Hank's (Full Strength)
Hank's Premix
Hank's Stock Solutions
Heat Shock (Diploid eggs)
In situ Hybridization Staining Buffer
Paramecia Seed Cultures
Paramecia for Baby Fish (Traditional Method)
Paramecia for Baby Fish (Streamlined Procedure)
Paramecia Medium Stock Solution I
Paramecia Medium Stock Solution II
Paramecia Storage Medium (Long Term)
PBS
2X PBS
PBS/BSA/DMSO
PCR Extraction Buffer
PMSF
PO4 buffer (0.1 M, pH 7.3)
Pronase
Protein Extraction Buffer
PTU
Ringer's Solutions
Salt Stock
SDS Sample Buffer
Schönefeld's Medium for Growing Paramecia
Sodium Bicarbonate
Sperm Freezing Medium
Sperm Thawing Medium
Stock salts
Subbing Solution for Slides
TBS
TES buffer
Tricaine
TTBS
Western Blot color development buffer
Western Blot color development solution
Whole-Mount in situ Hybridization Solutions

CHAPTER 10 - RECIPES



ABC Solution (Avidin-Biotin HRP Complex):


3ml 1% DMSO, 0.1% Triton, PBS
30µl Vectastain solution A
30µl Vectastain solution B


Agar/Sucrose:


1.5% agar
5% M sucrose
Boil into solution and store as 1.5 ml aliquots at 4°C.


Atabrine Stock Solution:


10 mg/ml dH2O. Store in a light tight bottle.


Blocking Solution for Western Blots:


3% dried milk in TBS

BSA/PBS/DMSO:


1% bovine serum albumin, 1% dimethyl
sulfoxide in PBS.

BT Fix:


10 ml 10% para-formaldehyde
15 ml 1.25X fix buffer
Adjust pH to 7.3 if necessary. Add dH2O to a final volume of 25.0 ml. Final concentration is 4% para-formaldehyde, 0.15 mM CaCl2, 4%sucrose in 0.1 M PO4 buffer.

Chorion Removal:


Drain eggs add 5 ml of 0.5 mg/ml pronase for 3.5 min.
Dilute eggs with 200 ml of 8x water. Rinse 3x more with 200 ml washes of 8x water.
8x water = 12 ml stock salts per liter dH2O
Stock salts = 40 g Instant Ocean per liter dH2O

DAB (Diaminobenzidine):


DAB is prepared by dissolving the contents of 1 g bottle of DAB in 25 ml dH2O and filtering with a disposable unit attached to a disposable syringe. Aliquots containing 1 mg DAB per 25 µl of solution are stored in 1.5 ml microfuge tubes in a -20°C freezer. Thawed aliquots may be refrozen. To minimize risk in handling this carcinogen, use disposable plastic containers and pipettes wherever possible. Treat waste with bleach.

DAB Heavy Metal Stain:


Presoak solution


4 mg DAB
5 ml 0.6% Ni(NH4)2(SO4)2
5 ml 0.2 M Tris buffer, pH 7.4
50 µl DMSO

After presoak interval add


10 µl 3% H2O2

Final concentrations are 0.04% DAB, 0.1 M Tris buffer, 0.3% Ni(NH4)2(SO4)2 and 0.003% H2O2.

DAB Presoak Solution:


25 µl (=1 mg) DAB stock solution
1 ml 0.1 M PO4 Buffer, pH7.3
1 ml dH2O
20 µl DMSO
After presoak interval add
5 µl 3% H2O2
Final concentrations are 0.05% DAB, 0.05 M PO4, 1% DMSO and 0.004% H2O2.

DAB Solution:


0.05% diaminobenzidine
1% dimethyl sulfoxide in 0.05 M PO4 buffer, pH 7.3.

DNA Extraction Buffer:


10 mM Tris pH 8.2
10 mM EDTA
200 mM NaCl
0.5% SDS
200 µg/ml proteinase K

EDTA:


Stock - 10 mM EDTA, pH 7.0. Add 1 ml of stock/10 ml Ringer's (final conc. 1 mM EDTA)

Egg Water:


1.5 ml stock salts added to 1 L distilled water = 60µg/ml final concentration.

Embryo Extract:


1. Chill 200 3 d embryos after removing from chorions.
2. Rinse in 0.5% chilled bleach for 2 min and then in zero calcium Ringer for 2 min.
3. Transfer to a Dounce homogenizer with a minimum of liquid and homogenize well.
4. Resuspend in 1 ml L-15 supplemented with 0.3 mg/ml glutamine, 50 U/ml penicillin, 0.05 mg/ml streptomycin and 0.8 mM CaCl2.
5. Store at -20°C.

Embryo Medium:


1.0 ml Hank's Stock #1
0.1 ml Hank's Stock #2
1.0 ml Hank's Stock #4
95.9 ml dd H2O
1.0 ml Hank's Stock #5
1.0 ml fresh Hank's Stock #6
Use about 10 drops 1 M NaOH to pH 7.2

Epon:


12 g Epon 812
24.7 g Dodecenyl succinic anhydride
Weigh components into a 50 ml plastic beaker.
Cover with parafilm and stir on a magnetic stirrer for
15 min. Add 0.5 ml DMP-30

Continue stirring for another 15 min. Store in syringes in -20°C freezer.

Epon/Araldite:


24.7 g Epon 812
33.25 g Dodecenyl succinic anhydride
31.05 g Araldite 506

Mix well by stirring. Use disposable syringes to add:
2.3 ml Dibutylphthalate
2.5 ml DMP-30

Continue to mix well by stirring. Try to avoid incorporating excess air. If this occurs resin may be degassed by subjecting it to a mild vacuum. Store resin in 10 ml syringes at -20°C.

Finquel (MS222):


See Tricaine. (Finquel is the trademark brand of Argent Chemical Laboratories, Inc.)

Fish Water:


60 mg "Instant Ocean" per liter dH2O.

Fix Buffer:


Dilute 1.25X fix buffer 3:2 with dH2O.

1.25X Fix Buffer:


1.0 g Sucrose
18.75 µl 0.2 M CaCl2
5 ml 0.5 M PO4 buffer, pH 7.3
Check pH. Adjust if necessary to 7.3 with 1 M NaOH or HCl.
Add H2O to a final volume of 15 ml.

Fixatives:


Fix buffer: 4% sucrose, 0.15 mM CaCl2, 0.1 M PO4 pH 7.3.
For general fixation: 1.5% glutaraldehyde, 0.5% paraformaldehyde in fix buffer.
For antibody staining: 4% paraformaldehyde in fix buffer.

Gelatin Embedding Medium:


17% gelatin in 10% Hank's saline.

Genomic DNA Extraction Buffer:


10 mM Tris pH 8
100 mM EDTA pH 8
0.5% SDS
200 µg/ml Proteinase K

GHCl Buffer:


7.5 M guanidinium hydrochloride
0.025 M NaOAc pH 7.0
5 mM dithiothreitol
0.5% N-laurylsarcosinate.

Giemsa:


4 mls Giemsa Stock (Sigma Diagnostics)
4 mls 0.5 M Na Phosphate pH7
200 mls distilled water

Ginzburg Fish Ringers:


6.5 g NaCl
0.25 g KCl
0.3 g CaCl2 (0.4 g CaCl2•2H2O)
Add ddH2O to almost 1 liter
0.2 g NaHCO3
Add ddH2O to 1 liter
Note: The order of addition is important to prevent precipitation

GIT Buffer:


4 M guanidinium isothiocyanate
0.1 M Tris-HCl pH 7.5
1% ß-mercaptoethanol.

Growth Medium:


L-15 (Sigma)
0.3 mg/ml glutamine
50 U/ml penicillin
0.05 mg/ml streptomycin
0.8 mM CaCl2
10% embryo extract
3% fetal calf serum.

Hank's (Final):


9.9 ml Hank's Premix
0.1 ml Stock #6

Hank's (Full Strength):


0.137 M NaCl
5.4 mM KCl
0.25 mM Na2H PO4
0.44 mM KH2 PO4
1.3 mM CaCl2
1.0 mM Mg SO4
4.2 mM NaH CO3

Hank's Premix:


Combine the following in order:
10.0 ml Solution #1
1.0 ml Solution #2
1.0 ml Solution #4
86.0 ml ddH2O
1.0 ml Solution #5

Store Hank's Premix in the refrigerator along with the Hank's solutions.

Hank's Stock Solutions:


Stock #1


8.0 g NaCl
0.4 g KCl
in 100 ml dd H2O

Stock #2


0.358 g Na2HPO4 Anhydrous
0.60 g KH2PO4
in 100 ml ddH2O

Stock #4


0.72 g CaCl2
in 50 ml ddH2O

Stock #5


1.23 g MgSO4x7H2O
in 50 ml dd H2O

Stock #6


0.35 g NaHCO3
10.0 mls dd H2O

Heat Shock (Diploid eggs):


T = 0 Fertilize eggs with UV-irradiated sperm
T = 5 min Transfer embryos to 28.5°C cylinder
T = 13 min Move cylinder from 28.5°C to 41.4°C
T = 15 min Move cylinder from 41.4°C to 28.5°C

In situ Hybridization Staining Buffer:


100 mM Tris pH 9.5
50 mM MgCl2
100 mM NaCl
0.1% Tween-20
1 mM Levamisol (add fresh)

Paramecia Seed Cultures:


1. Add 10-15 grains of boiled wheat to 175 mls of dH2O.
2. Inoculate with 20 ml from an excellent existing seed culture dish or with a sample from the commercial inoculant.
3. Grow for 7 to 12 days before using.

Paramecia for Baby Fish (Traditional Method):


1. Use glass finger bowls filled two-thirds full with system water.
2. To each bowl add 8-9 grains of of boiled wheat and 8 ml from a paramecia seed culture.
3. Stack the finger bowls 6 or 8 high, cover, and store at 28.5°C on well-lit shelves.
4.After 10-14 days, the paramecia are ready to feed to the fish larvae. Cultures remain useable and healthy for a month or more.

Paramecia for Baby Fish (Streamlined Procedure):


1. Fill plastic mouse cage or equivalent with 2 liters of system water.
2. Add a large pinch (about 40-50 grams) of boiled wheat, four 250 mg tablets of brewer's yeast, and 100 ml (or half the contents of a plastic seed culture dish) of paramecia culture to each mouse cage.
3. Cover and store in a warm, well-lit place. The covered mouse cages may be stacked three layers high with the highest layer closest to a warm light. It will be ready to feed first.
4. Brewer's yeast may also be added to the seed cultures (one half of a 250 mg tablet per 200 ml of seed culture). The cultures are then ready to use in four days and are depleted after two weeks.

Paramecia Medium Stock Solution I:


Component - g/l in dH2O - Source
Calcium pantothenate - 1 - Sigma P-2250
Nicotinamide - 1 - Sigma N-3376
Riboflavin - 1 - Sigma R-9881
Pyridoxamine HCL - 1.16 - Sigma P-9158
Folic Acid - 0.5 - Sigma F-7876
Thiamine HCL - 3 - Sigma T-4625
Biotin - 0.00125 - Sigma B-4639
(make 100x stock) Store at -20°C the riboflavin is dispersed through-out the solution and will settle to the bottom. Shake well before using

Paramecia Medium Stock Solution II:


Component - g/l in ETOH - Source
Stigmasterol - 2 - Sigma S-6126
Lipoic Acid (Dl-6,8-thioctic acid) - 0.02 - Sigma T-1395
Palmitic Acid - 3 - Sigma P-5917
Stearic Acid - 2 - Sigma S-4751
Oleic Acid - 0.4 - Sigma 0-4379
Linoleic Acid - 0.2 - Sigma L-1376
Linolenic Acid - 0.06 - Sigma L-2376
(Note: Blow nitrogen gas over the solution and store at -20°C)

Paramecia Storage Medium (Long Term):


Component - Stock - Amount (ml)
NaH2PO4 - 0.01 M - 10
Na2HPO4 - 0.01 M - 10
Sodium citrate - 0.1 M - 20
CaCl2 - 0.1 M - 15
dH20 - 945

PBS:


0.8% NaCl
0.02% KCl
0.02 M PO4, pH 7.3.

2X PBS:


8.0 g NaCl
0.2 g KCl
200 ml 0.1 M PO4 Buffer, pH 7.3
300 ml dH2O

When diluted 1:1 with dH2O, final concentrations are 0.8% NaCl, 0.02% KCl and 0.02 M PO4.

PBS/BSA/DMSO:


50 ml 2X PBS, pH 7.3
1 g BSA
1 ml DMSO

Check pH with paper and adjust if necessary. Add dH2O to a total volume of 100 ml

PCR Extraction Buffer:


10 mM Tris pH 8
2 mM EDTA
0.2% Triton X-100,
200 µg/ml Proteinase K

PMSF:


Stock - 100 mM phenylmethylsulfonylfluoride in isopropanol. Immediately before use, add 30 µl of stock/10 ml Ringer's (final conc. 0.3 mM PMSF).

PO4 buffer (0.1 M, pH 7.3):


77 ml 0.1 M NaH2PO4 (13.8 g NaH2PO4xH2O/liter dH2O)
23 ml 0.1 M Na2HPO4 (14.2 g Na2HPO4/liter dH2O)

Pronase:



5 mg/ml pronase diluted to 1 mg/ml in embryo medium

Protein Extraction Buffer:


10 mM tris, pH 7.4
2% Triton-X 100
1 mM PMSF
1 mM aprotinin
1 mM leupeptin
1 mM trypsin inhibitor

PTU:


0.003% 1-phenyl-2-thiourea in 10% Hank's saline.

Ringer's Solutions:

Normal


116 mM NaCl
2.9 mM KCl
1.8 mM CaCl2
5 mM HEPES, pH 7.2.

High calcium


116 mM NaCl
2.9 mM KCl
10 mM CaCl2
5 mM HEPES, pH 7.2.

Calcium free


116 mM NaCl
2.9 mM KCl
5 mM HEPES, pH 7.2.

Salt Stock:


20 tablespoons (280 g) Instant Ocean Sea Salts (Aquarium Systems, Inc.) dissolved in 2 liters distilled water

SDS Sample Buffer:


0.63 ml 1M Tris-HCl, pH 6.8
1.0 ml glycerol
0.5 ml ß-mercaptoethanol
1.75 ml 20% SDS
6.12 ml H2O
(10 ml total)

Store at -20°C in aliquots.

Schönefeld's Medium for Growing Paramecia:


Component - Concentration - Source
Powdered Skim Milk - 8.5 g/l - Any grocery store
Ribonucleic Acid - 1.0 g/l - Sigma R-6625
MgSO4 - 0.5 g/l - Sigma M-2643
Phosphatidylcholine - 250 mg/l - Sigma P-5638

Stock Solution I: 5 ml/l
Stock Solution II: 2.5 ml/l

Sodium Bacarbonate


0.35 g NaHCO3
10.0 ml ddH2O

Sperm Freezing Medium:


9 ml Ginzburg Fish Ringers
1 ml Methanol
1.5 g Carnation Powdered Skim Milk
Note: This order is important to prevent precipitation of milk

Sperm Thawing Medium:


a. Measure the mm of sperm + freezing medium (sm) in the capillary.
b. Convert to volume:
10µl (cap. vol.) x mm sm = vol (µl)
90 mm (cap. length) 1
c. Multiply the calculated volume x 10 and use that much 10% Hank's saline for thawing.

Stock Salts


40 g "Instant Ocean" Sea Salts added to 1 L distilled water

Subbing Solution for Slides:


0.5% gelatin
0.05% CrK(SO4)x12H2O.

TBS:


20 mM Tris, pH 7.5
500 mM NaCl

TES buffer:



1% sodium dodecyl sulfate
5 mM EDTA
10 mM Tris-HCl pH 7.4.

Tricaine:


Tricaine (3-amino benzoic acid ethyl ester also called ethyl 3-aminobenzoate) comes in a powdered form from Sigma (Cat.# A-5040). It is also available as Finquel (Part No. C-FINQ-UE) from Argent Chemical Laboratories, Inc. Make tricaine solution for anesthetizing fish by combining the following in a glass bottle with a screw cap:
400 mg tricaine powder
97.9 ml DD water
~2.1 ml 1 M Tris (pH 9).
Adjust pH to ~7. Store this solution in the freezer. (Buy the smallest amount possible because tricaine gets old.)
To use tricaine as an anesthetic combine the following in a 250 ml beaker:
4.2 ml tricaine solution
~100 ml clean tank water.

TTBS:


20 mM Tris, pH 7.5
500 mM NaCl
0.05% Tween-20

Western Blot color development buffer:


102 mg MgCl2
4.2 g NaHCO3
500 ml dH2O

Western Blot color development solution:


66 µl nitroblue tetrazolium (NBT) stock
33 µl 5-bromo-4-chloro-3-indolyl galactopyranoside
(BCIP) stock
10 ml color development buffer
Both NBT and BCIP stocks are 50 mg/ml in dimethylformamide. NBT stock is made by suspending 50 mg NBT in 700 ml dimethylformamide. Vortex. Add 300 µl distilled water to dissolve. Store both stocks at 4°C in dark.

Whole-Mount in situ Hybridization Solutions:



PBST - PBS plus 0.1% Tween
SSCT - SSC plus 0.1% Tween
HYB* - 50% formamide, 5xSSC, 0.1% Tween-20
HYB+ - HYB* with 5mg/ml torula (yeast) RNA, 50 µg/ml heparin