Nov 27, 2023

Public workspaceImmunohistochemistry Protocol for Paraffin-Embedded Sections - General

DOI
dx.doi.org/10.17504/protocols.io.kqdg3p7mql25/v1
  • 1Ottawa Hospital Research Institute;
  • 2OHRI
Julianna Tomlinson
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DOI: dx.doi.org/10.17504/protocols.io.kqdg3p7mql25/v1
Protocol Citation: Julianna Tomlinson, Nathalie Lengacher 2023. Immunohistochemistry Protocol for Paraffin-Embedded Sections - General. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3p7mql25/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
We use this protocol and it's working
Created: May 17, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 62744
Keywords: Immunohistochemistry Protocol, Antigen retrieval, ASAPCRN
Abstract
This protocol details general immunohistochemistry procedure.
Attachments
Icon for gegpbqbhp.pdf
gegpbqbhp.pdf
183KB
Materials
For DAB:
Solution-based: Sigma Cat# D5637; prepared in Tris buffer as per manufacturer's instructions
SIGMAFAST DAB Tablets: Sigma Cat# 4293
SIGMAFAST DAB with Metal Enhancer - SIgma Cat# 0426
Day 1 - Deparaffinize
Day 1 - Deparaffinize
Dip slides in 100% Citrisolv 3 x 5 mins.

Dip slides in 100% Citrisolv 3 x Duration00:05:00 (1/3).
5m
Dip slides in 100% Citrisolv 3 x Duration00:05:00 (2/3).
5m
Dip slides in 100% Citrisolv 3 x Duration00:05:00 (3/3).
5m
Hydration
Hydration
Dip in 100% EtOH 2 x 2 mins.

Dip in 100% EtOH 2 x Duration00:02:00 (1/2).
2m
Dip in 100% EtOH 2 x Duration00:02:00 (2/2).
2m
Dip in 95% EtOH 2 x 2 mins.
Dip in 95% EtOH 2 x Duration00:02:00 (1/2).
2m
Dip in 95% EtOH 2 x Duration00:02:00 (2/2).
2m
Dip in 80% EtOH 2 x 2 mins.

Dip in 80% EtOH 2 x Duration00:02:00 (1/2).
2m
Dip in 80% EtOH 2 x Duration00:02:00 (2/2).
2m
Dip in ddH2O 2 x 2 mins.

Dip in ddH2O 2 x Duration00:02:00 (1/2).
2m
Dip in ddH2O 2 x Duration00:02:00 (2/2).
2m
Quench endogenous peroxides
Quench endogenous peroxides
Incubate slides in 0.3% H2O2 in methanol for Duration00:15:00 (Amount500 µL of 30% H2O2 in Amount50 mL MetOH, in white containers).

15m
Incubation
Antigen retrieval
Antigen retrieval

Note
Use citrate buffer Ph6 or Tris-EDTA buffer Ph9 based on antigen desired.



Pre-heat antigen retrieval solution in microwave in white containers (~Amount50 mL ), for Duration00:05:00 at full power. Use large plastic container and fill halfway with water.

5m
Transfer slides to white containers with AR solution. Microwave for Duration00:10:00 at power level 1.

10m
Rest slides at TemperatureRoom temperature for Duration00:20:00 , change water in bottom of container.

20m
Wash
Wash
Remove AR solution and wash slides with ddH2O for Duration00:05:00 .

5m
Wash
Draw bubble with hydrophobic pen during this wash.
Wash
Rinse slides with PBS for Duration00:05:00 .

5m
Wash
Blocking
Blocking
Block with 5% normal goat serum in PBS + 0.1% Triton-x-100 + 0.05% Tween-20, Duration00:30:00 @ TemperatureRoom temperature (Amount300 µL /slide).

30m
Primary antibody
Primary antibody
Dilute antibody in 5% normal goat serum in PBS + 0.1% Triton-x-100 + 0.05% Tween-20, DurationOvernight at Temperature4 °C (Amount300 µL /slide).

5m
Day 2 - Wash
Day 2 - Wash
Rinse with PBS; 3 x 5 minutes
Wash
Rinse with PBS; 3 x Duration00:05:00 (1/3).
5m
Rinse with PBS; 3 x Duration00:05:00 (2/3).
5m
Rinse with PBS; 3 x Duration00:05:00 (3/3).
5m
Secondary antibody
Secondary antibody
Add secondary antibody diluted 1:225 in PBS + 0.1% triton-X + 0.05% tween-20 with 5% normal goat serum (~Amount300 µL /slide).

Pipetting
Commonly: biotinylated anti-rabbit IgG (H+L) made in goat (Vector Labs,BA-1000) or biotinylated anti mouse IgG (H+L) made in goat (Vector Labs, BA-9200).
Incubate at TemperatureRoom temperature for Duration01:00:00 - Duration02:00:00 .

3h
Incubation
Wash
Wash
Rinse with PBS; 3 x 5 minutes.
Wash
Rinse slides with PBS; 3 x Duration00:05:00 (1/3).
5m
Rinse slides with PBS; 3 x Duration00:05:00 (2/3).
5m
Rinse slides with PBS; 3 x Duration00:05:00 (3/3).
5m
Amplification
Amplification
Prepare amplification solution at least Duration00:30:00 before use.

30m
Amplification with VECTASTAIN® Elite® ABC HRP Kit.

Amount9 µL solution A per mL PBS, vortex; + Amount9 µL solution B per mL PBS, vortex; (Amount300 µL /slide).

Incubate at TemperatureRoom temperature for Duration01:00:00 -Duration02:00:00 .

3h
Incubation
Wash
Wash
Rinse slides with PBS; 3 x 5 minutes
Rinse slides with PBS; 3 x Duration00:05:00 (1/3).

5m
Rinse slides with PBS; 3 x Duration00:05:00 (2/3).
5m
Rinse slides with PBS; 3 x Duration00:05:00 (3/3).
5m
Substrate development
Substrate development

Note
Use DAB or eDAB depending on antigen being detected.


For regular DAB:
Add Amount1 mL of DAB to Amount150 mL of Concentration50 millimolar (mM) Tris +Amount50 µL 30% H202.

Pipetting
Dip slides into DAB solution, develop until colour appears ~ Duration00:03:00 .

3m
For eDAB (SIGMAFAST™ DAB with Metal Enhancer):
Add 1 tablet DAB/Cobalt and 1 tablet Urea Hydrogen Peroxide to Amount5 mL ddH2O, vortex until dissolved.


Note
Prepare immediately before use.

Pipetting
Use ~Amount300 µL /slide, develop until colour appears ~ Duration00:03:00 .

3m
Pipetting
Rinse with PBS to stop reaction.
Wash
Rinse with ddH20 for Duration00:05:00 .

5m
Wash
Counterstain
Counterstain
Counterstain with 1:2 Hematoxylin:ddH2O, dip slides in quickly.
Remove excess hematoxylin in ddH2O; 2X.
Dehydration
Dehydration
5 dips, 1 minute each:

Dip in 50% EtOH Duration00:01:00

1m
Dip in 80% EtOH x2 Duration00:01:00 .

1m
Dip in 95% EtOH Duration00:01:00 .

1m
Dip in 100% EtOH x2 Duration00:01:00 .

1m
Dip in 1:1 Citrisolv: 100% EtOH Duration00:01:00 .

1m
Dip in 100% Citrisolv x2 Duration00:01:00 .

1m
Coverslip
Coverslip
Coverslip with permount solution, allow slides to dry before viewing on microscope.