Sep 25, 2025
Immunofluorescence Staining of Brain Slices
- Tom Deerinck1
- 1UCSD
- NCMIR@UCSD
Protocol Citation: Tom Deerinck 2025. Immunofluorescence Staining of Brain Slices. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2o7y4v1y/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: February 21, 2022
Last Modified: September 25, 2025
Protocol Integer ID: 58491
Keywords: immunofluorescence staining, brain slices, UCSD, NCMIR, immunofluorescence staining of brain slices primary antibody, brain slices primary antibody, immunofluorescence staining, fluorescence secondary anitbody, fluorescence, specific protein, secondary anitbody
Funders Acknowledgements:
NIH/National Institute of Neurological Disorders and Stroke
Grant ID: U24NS120055
NIH/National Institute of General Medical Sciences
Grant ID: R24GM137200
Abstract
Primary antibody labeling to a specific protein using a fluorescence secondary anitbody to label it.
Troubleshooting
Rinse tissues with ice cold 1x PBS
3x @ 10 min
Make up blocking buffer
1x PBS, containing
3% normal donkey serum
1% bovine serum albumin
0.1% Triton X-100
Block tissues in blocking buffer 1hr on ice
Rinse tissues with ice cold working buffer
1x PBS, containing
10% Blocking Buffer
0.1~0.5% Triton X-100
Make double- or triple- 1stAbs cocktail in Working Buffer
Incubate with 1stAbs solution 24 to 72 hrs at 4°C
Rinse with ice-cold working buffer
6x @ 5 min
Dilute double- or triple- 2nd Abs cocktail in ice-cold working buffer
Incubate with 2nd Abs solution 2hr at 4°C
Rinse with 1x PBS
3x @ 5 min
Incubate tissues with DAPI or Hoechst 33342 solution for 5 minutes
Rinse with 1x PBS
3x @ 5 min
Mount cover slip using mounting medium