May 20, 2026

IF staining CD68

DOI
https://dx.doi.org/10.17504/protocols.io.rm7vzxw24gx1/v1
  • 1KU Leuven
Hanne HD Dhondt
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DOI: https://dx.doi.org/10.17504/protocols.io.rm7vzxw24gx1/v1
Protocol Citation: Eduard Bentea, Hanne Dhondt, Aisha Sati 2026. IF staining CD68. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzxw24gx1/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: January 16, 2024
Last Modified: May 26, 2026
Protocol  Integer ID: 96550
Keywords: staining cd68, month old mouse brain, old mouse brain, cd68, cd68 this protocol detail
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-024297
Abstract
This protocol details the CD68 IF staining of free floating sagittal sections from 2 month old mouse brains.
Attachments
Icon for Protocol_IF-CD68.docx
Protocol_IF-CD68.doc...
34KB
Guidelines
General note

  • Make sure that at each incubation step the tissue is flat and completely in touch with the liquid!!!
  • 488: green channel
  • 555: yellow/orange channel
  • 647: red channel
  • 405: blue channel

4 sections per animal



Materials
Materials
  • 24 well plates
  • oven (80 °C )
  • Ice
  • Wobbler
  • Aluminium foil
  • Falcon tubes (to prepare blocking and antibody dilutions)
  • Petri dish
  • Gelatine coated microscope slides
  • Cover slips #1.5 (Epredia; BB02400500SC13MNZ0)

Reagents
  • Citrate buffer 1 Molarity (M) 6
  • PBS (Gibco; 21600-096)
  • PBS 0.1 % Tergitol (ThermoScientific; 464252500; CAS: 68131-40-8)
  • Donkey serum (Jackson Immuno Research; 017-000-121; RRID: AB2337258)
  • CD68 primary antibody (Abcam; Ab 125212; RRID: AB_10975465)
  • Fluorescently labeled secundary antibody (Donkey anti-rabbit Alexa 488 (Abcam; Ab150073; RRID:AB_2636877) or Donkey anti-rabbit Alexa 647 (Abcam; Ab150075; RRID:AB_2752244))
  • Distilled water (AD)
  • Mowiol
  • DAPI

Day 1
ANTIGEN RETRIEVAL
Rinse the sections with PBS (Gibco; 21600-096).
Add 500 µL 1 Mass Percent Citrate buffer (6 ) in each well. Make sure the tissue is flat and completely in contact with the buffer!

00:30:00 incubation at 80 °C , 00:20:00 in the same buffer On ice .

50m
1X PBS rinse.
1X PBS rinse.
1X PBS 00:05:00 on the wobbler.

5m
250 µl blocking step: in 1 x PBS 0.1% Tergitol (ThermoScientific; 464252500; CAS: 68131-40-8) (PBS-T) + 10% Donkey serum (Jackson Immuno Research; 017-000-121; RRID: AB2337258) (accordingly to the secondary Abs) 00:40:00 incubation on the wobbler.

40m
250 µl Primary Ab CD68 (Abcam; Ab 125212; RRID: AB_10975465) 1:500 in 1 x PBS-T + 10% Donkey serum (accordingly to the secondary Abs) Overnight at Room temperature on the wobbler.

5m
Day 2
4h 10m
1 x PBS-T rinse.
1 x PBS-T 00:05:00 on shaker.

5m
1 x PBS-T 00:05:00 on shaker

5m
250 µl secondary Ab 2 h 1/500 in 1 x PBS-T 02:00:00 Room temperature on the wobbler (dark by covering with aluminium foil).

2h
1 x PBS rinse.
1 x PBS 00:05:00 on shaker.

5m
1 x PBS 00:05:00 on shaker.

5m
  • Briefly rinse sections in ½ PBS + ½ AD, mount them on a microscope slide (Avantor VWR Microscope slides cut edges frosted; 631-1551) and allow them to dry Overnight while covered.

2h
  • Cover with 80 µL mowiol and DAPI 1/500 (stains nucleus, 405 nm). Use cover slips #1.5 (Epredia; BB02400500SC13MNZ0). Let dry Overnight .

Day 3
4h 10m
Visualise the stained sections with Leica DM6 B LED Fluorescent microscope and the LasX software.
Quantify the images in FIJI
Protocol references
This protocol is kindly shared by María Sanchiz Calvo and Eduard Bentea of the Prof. Veerle Baekelandt lab.