CHAPTER 8 - HISTOLOGICAL METHODS
Solutions for Antibody Staining Protocols
(Source: R. BreMiller)
BT Fix:
10.0 ml 10% para-formaldehyde
15.0 ml 1.25X fix buffer
To make 25 mL of BT fix, combine 1 g sucrose, 18.75 µl .2M CaCl2,
5 mL 0.5 M phosphate buffer pH 7.3, 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% para-formaldehyde, 0.15 mM CaCl2, 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 1.25X fix buffer 3:2 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 ul of solution are stored in 1.5 ml microfuge tubes in a -20C 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 ul (=1mg) DAB stock solution
1 ml 0.1 M PO4 Buffer, pH7.3
1 ml dH2O
20 ul DMSO
After presoak interval add:
5 ul 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 ul DMSO
After presoak interval add:
10 ul 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 4C.
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 -20C.
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 -20C freezer.
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