Feb 05, 2024

Public workspaceImmunohistochemical staining, vibratome sections

DOI
dx.doi.org/10.17504/protocols.io.eq2lypnkqlx9/v1
  • 1KULeuven
Joris Van Asselberghs
Open access
DOI: dx.doi.org/10.17504/protocols.io.eq2lypnkqlx9/v1
Protocol Citation: Joris Van Asselberghs, Veerle Baekelandt 2024. Immunohistochemical staining, vibratome sections. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lypnkqlx9/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: November 07, 2020
Last Modified: May 31, 2024
Protocol Integer ID: 44295
Keywords: vibratome, sections, brain, neurological, IHC, staining, Immunohistochemical, ASAPCRN
Abstract
This protocol outlines general procedures for Immunohistochemical staining, using vibratome sections.
Attachments
Immunohystochemical_staining,_vibratome_sections.pdf
Immunohystochemical_...
139KB
Materials
Reagents Needed
  • 3% H2O2 in 1 x PBS
  • 1 x PBS 0.1% Triton X-100
  • 1 x PBS 0.1% Triton X-100 + 10% serum (accordingly to the secondary Abs)
  • Primary Antibodies (TH Ab152 1/5000)
  • Biotinylated Secondary Antibodies (SAR 1/300)
  • Streptavidine-HRP complex (1/1000)
  • DAB + H2O2 (1.4 µl H2O2 for 5 ml filtered DAB solution)
• DAB solution: 10 mg DAB (=1 tablet) for 25 ml 0.05 TRIS (TBS) pH 7.6; dissolve and filter through 0.22 µm filter, add H2O2 just before
  • PBS or PBS + 0.1% Na Azide
  • ½ PBS + ½ AD
  • Ethanol:
• 70% ethanol
• 90% ethanol
• 100% ethanol
  • Histoclear II
  • DPX mountant

Consumables Needed
  • Coverslips
  • Slides

Equipment Needed
  • Wobbler
Safety warnings
Attention
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Before start
This protocol uses floating sections in 24-well plate in Concentration1 X PBS .
Quench Endogenous Peroxidase Activity
Quench Endogenous Peroxidase Activity
20m
Incubate samples in 1 x PBS 3% H2O2, for Duration00:10:00 at TemperatureRoom temperature on the wobbler (to quench endogenous peroxidase activity) using Amount500 µL / well.

10m
Incubation
Rinse with Concentration0.1 % (v/v) Triton X-100 in 1x PBS .

Wash
Rinse with Concentration0.1 % (v/v) Triton X-100 in 1x PBS for Duration00:05:00 on wobbler.

5m
Wash
Repeat: rinse with Concentration0.1 % (v/v) Triton X-100 in 1x PBS for Duration00:05:00 on wobbler.

5m
Wash
Primary Antibody Incubation
Primary Antibody Incubation
40m
Add Amount250 µL Primary Abs in 1 x PBS 0.1% Triton X-100 + 10% serum (accordingly to the secondary Abs) DurationOvernight at TemperatureRoom temperature on wobbler.
Note
Dilution: 1st AB: TH Ab152 1/5000

40m
Incubation
Overnight
Rinse with Concentration0.1 % (v/v) Triton X-100 in 1x PBS .
Wash
Rinse with Concentration0.1 % (v/v) Triton X-100 in 1x PBS for Duration00:05:00 on wobbler.
5m
Wash
Repeat: rinse with Concentration0.1 % (v/v) Triton X-100 in 1x PBS for Duration00:05:00 on wobbler.
5m
Wash
Secondary Antibody Incubation
Secondary Antibody Incubation
20m
Add Amount250 µL biotinylated Secondary Abs in 1 x PBS 0.1% Triton X-100 for Duration00:30:00 at TemperatureRoom temperature on wobbler.
Note
Dilution: 2nd AB: SAR 1/300

30m
Incubation
Rinse with Concentration0.1 % (v/v) Triton X-100 in 1x PBS .
Wash
Rinse with Concentration0.1 % (v/v) Triton X-100 in 1x PBS for Duration00:05:00 on wobbler.
5m
Wash
Repeat: rinse with Concentration0.1 % (v/v) Triton X-100 in 1x PBS for Duration00:05:00 on wobbler.
5m
Wash
Streptavidine-HRP complex Incubation
Streptavidine-HRP complex Incubation
30m
Add Amount250 µL Streptavidine-HRP complex 1/1000 (in 1 x PBS 0.1% Triton X-100) for Duration00:30:00 at TemperatureRoom temperature on wobbler.
Note
Dilution: STRP-HRP 1/1000

30m
Incubation
Rinse with Concentration0.1 % (v/v) Triton X-100 in 1x PBS .
Wash
Rinse with Concentration0.1 % (v/v) Triton X-100 in 1x PBS for Duration00:05:00 on wobbler.
5m
Wash
Repeat: rinse with Concentration0.1 % (v/v) Triton X-100 in 1x PBS for Duration00:05:00 on wobbler.
5m
Wash
DAB + H2O2
DAB + H2O2

Safety information
Work on DAB Bench!

Prepare DAB solution: Amount10 mg DAB (=1 tablet) for Amount25 mL 0.05 TRIS (TBS) pH 7.6 ; dissolve and filter through 0.22 µm filter, add H2O2 just before use! Or Vector SG (TH).
(Add Amount1.4 µL H2O2 for Amount5 mL filtered DAB solution ).
Add Amount250 µL DAB + H2O2 (ON DAB BENCH!). Allow reaction to proceed for a few minutes at TemperatureRoom temperature without wobbling.
Rinse with Concentration0.1 % (v/v) Triton X-100 in 1x PBS .

Wash
Replace TBS Triton X-100 with PBS or Concentration0.1 % (v/v) Sodium Azide in PBS in case sections are not to be mounted immediately. Store at Temperature4 °C .
½ PBS + ½ AD Rinse
½ PBS + ½ AD Rinse
30m
Briefly rinse sections in ½ PBS + ½ AD and mount on gelatin coated microscopy slides. Allow to dry for Duration00:30:00 in flow or couple of hours on bench.

30m
Dehydration
Dehydration
25m
Dehydrate samples in Concentration70 % (v/v) ethanol for Duration00:05:00 .
5m
Dehydrate samples in Concentration90 % (v/v) ethanol for Duration00:05:00 .
5m
Dehydrate samples in Concentration100 % (v/v) ethanol for Duration00:05:00 .
5m
Repeat: dehydrate samples in Concentration100 % (v/v) ethanol for Duration00:05:00 .
5m
Replace ethanol with Histoclear II for Duration00:05:00 .
5m
Mounting
Mounting
30m
Mount coverslips on top of the slides with DPX and allow to dry DurationOvernight in the flow.
30m
Overnight
Press out bubbles next morning.