1. Modified PBS
• potassium phosphate, dibasic, anhydrous (K2HP04), 5.44 g
• bring volume to 1 litre with dH20 and adjust pH to 7.6 with 10 N NaOH.
2. 0.02 M Citrate buffer
• solution A: 0.01 M citric acid (MW 192.1), monohydrate (Sigma, C-0759), 0.192 g in 100 ml dH20
• solution B: 0.01 M citric acid (MW 294.1), trisodium salt (Sigma, C-8532), 0.294 g in 100 ml dH20
• adjust pH to 3.2 using concentrated HC1
• bring total volume to 1 litre
• sodium chloride 32.0 g=550 mM final
• potassium chloride 0.8 g=10 mM final
• add dH20 to 950 ml and adjust to pH 7.6-7.8 with HC1
• adjust total volume to 1 litre
6. Diaminobenzidine/H202 solution
Caution. DAB is a carcinogen. Use precautions and dispose of properly.
• dissolve diaminobenzidine in 0.1 M Tris (pH 8.1) at 0.5 mg/ml [3,3'-diaminobenzidine from Sigma powder (cat. no. D-5637) or in tablet form (D-5905)]. The final pH should be -7.6-7.8
• add 41 |xl of 30% H202 per 50 ml of DAB solution
• load into a 60 cm3 syringe
• filter the DAB on to the glass slides
• H202 should be purchased anew every 2-4 weeks
• do not add the H202 to the DAB solution until just before applying on to the slides
7. Methyl green counter stain
• add 1 g methyl green dye to 200 ml 50 mM acetate buffer (pH 5.5) in a glass container
• add —50 ml chloroform and shake vigorously
• allow layers to separate
• discard bottom (chloroform) layer
• repeat chloroform extractions until all traces of methyl violet (purple colour) have disappeared
• allow the methyl green staining solution to stand in an open flask overnight so that any residual chloroform evaporates
• if crystals remain, filter through Whatman 3MM paper
• the solution is stable for several months at room temperature
• methyl green can be reused several times. Filter through Whatman 3MM paper or equivalent
• prepare a 0.2 M acetic acid solution using glacial acetic acid (Fisher Sci., Inc.; cat. no. A38-212). Add 11.55 ml glacial acetic acid to 988.45 ml dH20.
• prepare a 0.2 M sodium acetate solution. Dissolve 27.2 g sodium acetate (Sigma; cat. no. S-8625) into 1 litre dHzO.
• dissolve 2.1 g citric acid (anhydrous) (MW 192) in 1 litre of dH20
The deparaffinization solutions (xylene, EtOH, citrate buffer) may be used for 2-4 weeks, depending upon the frequency of experimental procedures. The 2% H202 in methanol should be freshly prepared every two weeks. PBS should be prepared fresh each week. DAB should be made fresh each time.
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