Oct 25, 2022

Immunostaining on Paraffin Sections of Fly Heads V.2

DOI
https://dx.doi.org/10.17504/protocols.io.81wgby61nvpk/v2
  • 1Brigham and Women's Hospital;
  • 2Harvard Medical School
  • Daniel's workspace
Daniel El Kodsi
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DOI: https://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, paraffin sections of fly head, fly head, paraffin section, head, protocol
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 42 °C Overnight .

Deparaffinize and bring through ethanols to water (xylene x 2, 100% ethanol x 4, tap H2O x 2).
Microwave slides in 10 millimolar (mM) sodium citrate for 00:15:00 .
Cool 00:20:00 .

Stock: 100 millimolar (mM) sodium citrate, pH 6.0
Use at least 1 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 00:30:00 to 1h
Stock: 10X PBS
30m
Incubate with primary antibody Overnight at Room 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 01:00:00 at Room temperature .
For immunohistochemistry incubate in ABC reagent (Vector) for 01:00:00 at Room 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.