This document provides a general protocol for detecting streptavidin in fixed paraffin tissue sections using immunohistochemistry. The protocol involves deparaffinizing the tissue, permeabilizing it, blocking non-specific binding, incubating with primary and secondary antibodies, labeling with streptavidin conjugates, counterstaining if needed, and mounting for analysis. Several washes and blocking steps are included to reduce background signal.

1. General Labeling Protocol for Streptavidin Detection
For Fixed Paraffin Tissue Sections
Jason A. Kilgore, Molecular Probes Tech Support
Very thick sections may require longer wash and incubation times.
Tissue sections, particularly paraffin, may have autofluorescence issues that can lower
the signal-to-background ratio necessary to detect the antigen.
Primary antibodies of different species, or conjugated primary antibodies, may be mixed
together in one solution. Secondary antibodies recognizing different species or isotypes
of primary may be mixed together into one solution.
Some primary antibodies may require antigen retrieval.
All steps usually at RT, unless otherwise noted.
PBT = phosphate buffered saline (PBS) with 0.2% Triton X-100 and 0.1% bovine serum
albumin (BSA)
1) Deparaffinize in solvent. Histoclear or other citric solvent will have less
autofluorescence generation than xylenes
2) Rehydrate through a graded ethanol series back down to PBS
3) Permeabilize the section for at least 30 minutes (a common permeabilization
reagent is 0.4% Triton X-100 in PBS)
4) Wash well in PBT (typically 3 x 10 minutes)
5) (optional: Reduce autofluorescence using sodium borohydride: wash 3 x 10
minutes in 1mg/ml Na borohydride in PBS, each made IMMEDIATELY before
use)
6) Wash very well 3 x 10 minutes PBT
7) (optional: perform antigen retrieval method of choice; not all primary antibodies
require this, and it can increase autofluorescence)
8) (optional: Block for non-specific dye binding using the Image-iT FX Image
Enhancer Solution, I36933, for 30 minutes to 1 hour)
9) Block for non-specific antibody binding at least 60 minutes (a common blocking
solution would be 3-6% bovine serum albumin / 5% normal goat serum / PBS, or
commercial blocking reagents like our BlockAid, product B10710)
10) Incubate in primary antibody for at least 2 hours, in blocking solution (antibody
concentrations vary, but usually between 0.5-10ug/mL). Very commonly
incubation will go overnight at 4ºC.
11) Wash well in PBT
12) (steps 7 and 8 can be skipped if the primary is biotinylated) Incubate in
biotinylated secondary antibody for at least 2 hours, in 3-6% bovine serum
albumin / PBT (a good starting antibody concentration is 5 ug/mL)
13) Wash well in PBT

2. 14) Label with conjugated streptavidin 1 – 2 hours (2-5 ug/ml in 3-6% bovine serum
albumin / PBT )
15) Wash well in PBS
16) Counterstain as needed (such as with Qnuclear Deep Red Stain, Q10363)
17) Wash 3 x 10 minutes PBS
18) Mount in Qmount mounting media (Q10336) with required dehydration series
beforehand (seem Qmount manual)