Sep 11, 2025

Public workspaceImmunhistochemistry (Stadelmann Lab - single antibody, DAB-POX-Avidin-Biotin)

DOI
https://dx.doi.org/10.17504/protocols.io.8epv5rnw6g1b/v1
  • Jonas Franz1,
  • Katja Schulz1,
  • Christine Stadelmann1
  • 1University Medical Center Göttingen, Neuropathology
Jonas Franz
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DOI: https://dx.doi.org/10.17504/protocols.io.8epv5rnw6g1b/v1
Protocol Citation: Jonas Franz, Katja Schulz, Christine Stadelmann 2025. Immunhistochemistry (Stadelmann Lab - single antibody, DAB-POX-Avidin-Biotin). protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5rnw6g1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
This protocol is established over years and provides robust results.
Created: June 27, 2024
Last Modified: September 11, 2025
Protocol Integer ID: 102505
Keywords: ASAPCRN, primary antibody, single antibody, secondary antibody, coupled secondary antibody, using avidin, immunhistochemistry, avidin, biotin, coupled peroxidase, stadelmann lab
Abstract
This protocol describes staining of any primary antibody using a biotin coupled secondary antibody using Avidin coupled peroxidase.
Troubleshooting
Paraffin removal and rehydration
14h 35m
Immunohistochemistry was performed on 2-3 µm thick paraffin sections.
Remove Excess Paraffin at 56° for 1h-24h
1h
Rehydration
4x Xylol à 5 min
20m
1x Isopropanol:Xylol 1:1 à 3 min
3m
2x 100% Isopropanol à 3 min
6m
1x 90% Isopropanol à 3 min
3m
1x 70% Isopropanol à 3 min
3m
1x 50% Isopropanol à 3 min
3m
3x wash in aqua dest
1m 30s
Antigen Retrieval
14h 35m
Pretreatment
heat antigen retrieval (1x citrate buffer 10 mM , pH 6) - 30 min steamer

adopt according to tissue and antibody
3x wash PBS
formic acid (98%) (for Amyloid beta immunhistochemistry) - 1minute at RT
3x wash PBS
3% hydrogen peroxide in PBS 10 min @ RT
3x wash PBS
Blocking
14h 35m
Blocking in 10% normal goat serum in PBS for 10 min @ RT
10m
Decant 10% NGS in PBS and move directly to the next step without washing
Staining
14h 35m
Incubation of primary antibodies over night with respective dilution in 10% NGS @RT in a humid chamber


target antigen - humanhost speciesCloneCompanyCatalog NumberDilution
pTaumouseat8Thermo Fisher ScientificMN10201:100
Selected Antibodies

12h
Overnight
3x wash with PBS
1m 30s
secondary antibody
Incubation with secondary antibody coupled to biotin at RT for 1h in a humid chamber
target antigenhost speciesCloneCompanyCatalog NumberDilution
mouse IgGsheepGE HealthcareRPN10011:200
s

1h
3x wash PBS
1m 30s
ExtrAvidin-peroxidase 1:1000 in 10% NGS for 1h in humid chamber at RT
targetcoupled enzymeCompanyCatalog NumberDilution
biotinPeroxidase1:1000
1h
Counterstaining + Mounting
14h 35m
3x wash PBS
1m 30s
Slides were developed using DAB (1gin40mL PBS -> 1mL)
1 mL Aliquots + 50 mL PBS + 20 µL 30% H2O2)
10m
3x PBS

or optional
3x TBS -> move to AP coupled secondary staining step 9
(dx.doi.org/10.17504/protocols.io.3byl4952rgo5/v1 )
1m 30s
10 min tap water
10m
Hematoxylin 30s

30s
Differentiation dip in HCL
5s
Dehydration reverse see step 3
40m
Entellan mounting with coverslip