Solutions For Antibody Staining Protocols

(Source: R. BreMiller from Zebrafish Book 5th Edition)

BT Fix:

10.0 ml 10% paraformaldehyde
15.0 ml 1.25x fix buffer

To make 25 ml of BT fix, combine 1 g sucrose, 18.75 μl 0.2M CaCl2, 5 ml of 0.5 M phosphate buffer pH 7.3 and 10 ml 10% paraformaldehyde. Adjust the pH of the solution with 1M NaOH or HCl before bringing it to its final 25 ml volume with dH2O. Final concentration is 4% paraformaldehyde, 0.15 mM CaCl2 and 4% sucrose in 0.1 M PO4 buffer.

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 dH2O to a final volume of 15 ml.

Fix buffer:

Dilute 2x fix buffer 1:1 with dH2O.

PO4 buffer (0.1 M, pH 7.3):

80 ml 0.1 M Na2HPO4 (13.8 g Na2HPO4⋅H2O/liter dH2O)
20 ml 0.1 M NaH2PO4 (26.8 g NaH2PO4⋅7H2O/liter dH2O)

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

Diaminobenzidine (DAB):

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 presoak solution:

25 µl (=1mg) DAB stock solution
1 ml 0.1 M PO4 Buffer, pH 7.3
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 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.

Gelatin Embedding Medium:

17% gelatin in 10% Hank's saline.

Agar/Sucrose:

1.5% agar, 5% M sucrose, boiled into solution and stored as 1.5 ml aliquots at 4°C.

Subbing Solution for Slides:

1.5% gelatin, 0.15% CrK(SO4)⋅2H2O.

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.

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 minutes. Add 0.5 ml DMP-30

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