Aug 22, 2025

Public workspaceFree Floating IHC Fluorescent Staining 

DOI
https://dx.doi.org/10.17504/protocols.io.e6nvwd8qwlmk/v1
  • Ashley Harms1,
  • Jhodi Webster1
  • 1University of Alabama at Birmingham
Jhodi Webster
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DOI: https://dx.doi.org/10.17504/protocols.io.e6nvwd8qwlmk/v1
Protocol Citation: Ashley Harms, Jhodi Webster 2025. Free Floating IHC Fluorescent Staining . protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwd8qwlmk/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: October 20, 2023
Last Modified: August 22, 2025
Protocol Integer ID: 89662
Keywords: ASAPCRN, floating ihc fluorescent staining, ihc fluorescent staining, fluorescent labelling, sections for fluorescent labelling, floating section, floating ihc fluorescent, ihc fluorescent
Abstract
This protocol is optimized for to stain free floating sections for fluorescent labelling. Please note than each step must be done on a shaker.
Troubleshooting
Day 1
Using a 12-well plate with buckets, wash sections 3x10min TBS
Depending on antibody of choice, do an antigen retrieval step.
Antigen retrieval in TBS:    
10mM sodium citrate     
0.05% Tween-20
Block in 5% serum in TBST for 1hour at RT (using approx 2mL per well)
Using approx 500uL per well, let sections wash in primary antibody solution in TBST + 1% serum overnight in Temperature4 °C

Day 2
Wash sections 3x10min in TBST

Incubate in secondary antibody (1:1000 for Invitrogen-brand or 1:2000 for BioRad-brand in TBST + 1%serum) for 2 hours @ RT in the dark. Approx 2mL per well.
Wash 3x10min in TBS
Mount and allow slides to dry. Coverslip once sections are completely dried on the slide (.