Oct 25, 2022

Public workspaceImmunostaining on Paraffin Sections of Fly Heads V.2

DOI
dx.doi.org/10.17504/protocols.io.81wgby61nvpk/v2
  • 1Brigham and Women's Hospital;
  • 2Harvard Medical School
Daniel El Kodsi
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DOI: dx.doi.org/10.17504/protocols.io.81wgby61nvpk/v2
Protocol Citation: Mel Feany 2022. Immunostaining on Paraffin Sections of Fly Heads. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgby61nvpk/v2Version created by Daniel El Kodsi
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: October 25, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 71787
Keywords: ASAPCRN
Abstract
This protocol describes how to perform immunostaining on paraffin sections of fly heads.
Fix fly heads overnight in 4% formalin.
Embed in paraffin.
Cut 4 micron sections.  Dry at Temperature42 °C DurationOvernight .

Deparaffinize and bring through ethanols to water (xylene x 2, 100% ethanol x 4, tap H2O x 2).
Microwave slides in Concentration10 millimolar (mM) sodium citrate for Duration00:15:00 .
Cool Duration00:20:00 .

Stock: Concentration100 millimolar (mM) sodium citrate, pH 6.0
Use at least Amount1 L of citrate solution in large glass box to avoid drying.





35m
Block in PBST (PBS with 0.3% Triton) with 2% dry milk for Duration00:30:00 to 1h
Stock: 10X PBS
30m
Incubate with primary antibody DurationOvernight at TemperatureRoom temperature .

Wash 3 x in PBST.
Incubate with appropriate biotinylated secondary (for immunohistochemistry) or fluorescent secondary (for immunofluorescence) antibody at 1:200 in PBST + milk for Duration01:00:00 at TemperatureRoom temperature .
For immunohistochemistry incubate in ABC reagent (Vector) for Duration01:00:00 at TemperatureRoom temperature .

2h
Rinse 3 x PBST.
Mount slides with antifading medium for immunofluorescence or dehydrate through ethanol series and xylenes and mount in Permount for immunohistochemistry.