Reagents & Solutions

AnnV buffer
RBC Lysing solution
HRP development solution (AEC)
AP Developing solution
Blocking solutions.

Endogenous peroxidase blocking solution
Non-specific antibody background blocking solution: swine serum
Non-specific antibody background blocking solution: milk

Enzymatic antigen retrieval solution
Unconjugated, biotin- and fluorochrome-conjugated antibody (primary) solutions
Unconjugated, biotin- and fluorochrome-conjugated antibody (secondary) solutions
Enzyme-conjugated secondary antibody solutions.

Links

10 mM HEPES/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂.

 Ammonium chloride (NH₄Cl) 0.15M  8.29 g
 Potassium bicarbonate (KHCO₃) 10mM  1 g
 EDTA  0.1mM     0.037 g
 H2O dist      1 l
 filtered with 0.45µm filter, stored at RT.

HRP development solution (AEC):

For 50 ml developing solution add in order:

Aminoethylcarbazole (20 mg tablets, Sigma A-6926, dissolved in 2.5 ml NN-DM formamide)

50 ml acetate buffer pH 5.5 (52.5 ml of 0.1M acetic acid solution + 196.5 ml of a 0.1M Na acetate solution, bring to 500 ml)

25 µl H₂O₂ 30%.

Shake well
Filter with a 45µm filter (optional).
Keep away from direct light, use within 5 min.

For 50 ml developing solution add in order:

• 50 ml Tris-Hcl 0.1M pH 9.2 (exact!) (1:10 from a stock solution 1M)
• Levamisole 1mM (12 mg/50 ml) (Sigma L9756)
• 20 mg Naphtol As BI phosphate (Sigma N2250, the cheapo) (stock solution 40 mg/ml in NN-DM formamide, anhydrous, 0.5 ml aliquots kept at -20°C)
• 10 mg Fast Blue BB Diazonium (Sigma F3378)  salt or Fast Red Diazonium salt (Sigma F-6760)
Shake well
Filter with a 45µm filter.
Keep away from direct light, use within 5 min.

Blocking solutions.

Endogenous peroxidase blocking solution (H₂O₂-Azide)

H₂O₂ 3%, NaN₃ 1% in PBS 30 min.
Ref.
This method is preferred to 1.5% H₂O₂ in absolute methanol for 30 min.)

Non-specific antibody background blocking solution: swine serum.

Swine serum 1% in PBS (P9783, SIGMA.)
Use for all primary antibodies, except porcine.

Non-specific antibody background blocking solution: MILK.

skim milk 5% in TBS-T for 10-30 min. (Miller RT et al, Appl. Immunohist & Molecular Morphology, 71(1): 63-65, 1999) NOTE: skim milk may contain weak proteases which will be useful to further unmask antigens, but may reduce immunostaining by acting on primary Abs.

PBS   100ml
2M NaOH   4 drops
Trypsin   0.1g
CaCl₂   0.1g

Bovine Serum Albumin 1%
Sodium Azide (NaN₃) 0.1%.
PBS
The solution should be clear. Store at 4*C.
Do not use this solution to dilute HRP-containing reagents. NaN₃ will inactivate them.

Bovine Serum Albumin 1%
Swine serum 1%
Human serum 1%
Sodium Azide (NaN₃) 0.1%
PBS
The solution should be clear. Store at 4*C.
Do not use this solution to dilute HRP-containing reagents. NaN₃ will inactivate them.

Enzyme-conjugated secondary antibody solutions.

HRP-conjugates (use within the same day, the discard)

PBS
Bovine Serum Albumin 1% azide-free
Swine serum 1% azide-free
Human serum 1% Azide-free

AP-conjugates (use within the same day, the discard)

Bovine Serum Albumin 1%
Swine serum 1%
Human serum 1%
Sodium Azide (NaN₃) 0.1%
TBS