IHC Standards & Protocols

Lab Default Solutions and Procedures

All Immuno runs use the following, unless otherwise noted:

• 50 mM TBS, pH 7.2
• LTBS = 0.1% Triton X-100 + 3% Normal Donkey Serum (NDS) in 50 mM TBS, pH 7.2
• Triton: 0.1% = 1 µl/ml = 1:1k dilution. Triton Stock is made at 25% Triton, 75% TBS, so 0.1% now = 4 µl/ml = 4: 1k = 1:250 dilution. For 30 ml LTBS, use 120 µl of 25% Triton = 0.1%; for 50 ml, use 200 µl; for 100 ml, use 400 µl, and so on.
• All rinses (including sections transferred from cryo) are a minimum of 5 rinses of 5 min each. Records should indicate at minimum the total number of rinses and the total time rinsing.
• DAB reactions use 1 aliquot DAB (0.5 ml, 30mg/ml), 4.5 µl 30% H2O2 for 4.5 min; Reactions are stopped with 3 quick changes of TBS, followed by additional rinses before transferring sections to 6-well trays for mounting

IHC standards should be recorded in the notes, while any variant must be recorded

Dilution and Aliquoting of Streptavidin Conjugates

Single-Label IHC Protocol (Fluorescence & DAB)

Fluorescence Double Labeling (SAvidin & ∝FITC)