Jan 29, 2022

Public workspaceWhole mount immunohistochemistry (WM-IHC)

DOI
dx.doi.org/10.17504/protocols.io.b2xfqfjn
  • 1University of Fribourg
  • Blanchoud lab, UNIFR
Marta Wawrzyniak
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DOI: dx.doi.org/10.17504/protocols.io.b2xfqfjn
Protocol Citation: Marta Wawrzyniak, Nathalie Weber, Simon Blanchoud 2022. Whole mount immunohistochemistry (WM-IHC). protocols.io https://dx.doi.org/10.17504/protocols.io.b2xfqfjn
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: December 16, 2021
Last Modified: January 29, 2022
Protocol Integer ID: 56007
Keywords: colonial tunicates, WM-IHC, immunohistochemistry, ascidians
Abstract
This protocol has been successfully used with colonial tunicates has been adapted to these animals based on the following publication:

The onset of whole-body regeneration in Botryllus schlosseri: morphological and molecular characterization.
Materials
4% PFA
PBST (PBS + 0.1% Triton X-100)
PBST-SC (PBST + 3.9mM Sodium citrate)
PBST-GS (PBST + 5% Goat Serum)
primary and secondary antibobies
mounting medium
PAP pen
10x PBS, pH 7.2 (store for up to 2 moths at RT)

ABC
Na2HPO4*2H2O0.1M17.8g
KH2PO418mM2.4g
NaCl1.4M80g
KCl27mM2g
water1000mL
10x PBS (amounts calculated for 1L)


ABC
1x PBS50mL
Triton X-1000.1%50uL
Sodium citrate3.9mM50mg
PBST-SC (amounts calculated for 50mL)






Fixation
Fixation
1h 30m
1h 30m
Clean the slide with the colony of your interest.
Cleaning colonial ascidians.
Anesthetize the animals following this protocol. See Whole colony fixation V.1.


Fix the animals in 4% PFA DurationOvernight at TemperatureRoom temperature . See Whole colony fixation V.1.



Wash twice in 3.3x PBS for Duration00:30:00 .
30m
Rinse quickly 3 times with 1x PBS and then leave in 1x PBS for Duration01:00:00 .
1h
Permeabilization and quenching
Permeabilization and quenching
1h
1h

Place samples in PBST-SC.

Shake DurationOvernight at TemperatureRoom temperature .



Rinse once with 1x PBS.
Blocking and staining
Blocking and staining
3d 21h
3d 21h
Place samples in PBST-GS.
Shake at TemperatureRoom temperature for Duration02:00:00 .

2h
Stain your samples with primary antibody for Duration30:00:00 .

1d 6h
Dilute the primary antibody in PBST-GS according to the manufacturer.
On the slide, circle the system of interest with a PAP pen and wait Duration00:02:00 for the circle to dry.

2m
Place the slide flat into the humidity chamber.
Pipette the primary antibody solution on the circled system and incubate at Temperature4 °C for Duration30:00:00 .

1d 6h
Wash four times in 1x PBST at TemperatureRoom temperature for Duration00:30:00 slowly on a linear.

30m

Stain your samples with secondary antiboby for Duration30:00:00 .
1d 6h
Dilute the antibody in PBST-GS according to the manufacturer (keep solution and sample protected from the light!).
Incubate DurationOvernight in darkness at Temperature4 °C in the humidity chamber.

Wash four times in 1x PBST at TemperatureRoom temperature for Duration00:30:00 slowly on a linear shaker.
30m
Dry as much as possible.
Circle the sample with vaseline to build a custom-made chamber.
Mount with mounting medium, cover with a coverslip.
Image your slides directly or store them at Temperature4 °C .