Jan 15, 2026
Immunohistochemistry for astrocytes, microglia, and c-Fos
- Mario Alberto Bautista-Carro1
- 1Cinvestav
External link: https://doi.org/10.1371/journal.pone.0339028
Protocol Citation: Mario Alberto Bautista-Carro 2026. Immunohistochemistry for astrocytes, microglia, and c-Fos. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl46de8go5/v1
Manuscript citation:
Bautista-Carro MA, Sánchez-Teoyotl P, Juárez-Serrano D, Iannitti T, Díaz A, Flores G, Morales-Medina JC (2026) Olfactory bulbectomy induces neurobiological alterations in the prefrontal cortex and hyperlocomotion in male rats. PLOS One 21(1). doi: 10.1371/journal.pone.0339028
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 07, 2025
Last Modified: January 15, 2026
Protocol Integer ID: 229265
Keywords: Immunohistochemistry, Perfusion and fixation, GFAP (astrocytes), Iba1 (microglia), c-Fos (neuronal activity), fos performing immunohistochemistry for astrocyte, immunohistochemistry for astrocyte, microglia, astrocyte, glial fibrillary acidic protein, fos performing immunohistochemistry, immunohistochemistry, glial response to damage, glial response, marker of neuronal activity, performing immunohistochemistry, inflammation, neuronal plasticity, neuronal activity, ionized calcium
Abstract
Performing immunohistochemistry for astrocytes and microglia allows us to study changes in their morphology or cell density, which reflects glial response to damage, inflammation, or neuronal plasticity. Immunohistochemistry for c-Fos is used as a marker of neuronal activity induced by specific stimuli, behaviors, or experiences. Below we describe a protocol for performing immunohistochemistry for astrocytes [glial fibrillary acidic protein (GFAP)], microglia [ionized calcium-binding adaptor molecule 1 (Iba1)], and c-Fos.
Guidelines
It is important that the sections are stirred continuously during incubation.
Materials
Vibratome, 1x phosphate buffer solution, hydrogen peroxide (H₂O₂), normal rabbit serum, normal goat serum, primary antibodies (goat anti-GFAP ab53554, rabbit anti-Iba-1 cell signaling), secondary antibodies (Goat IgG VECTASTAIN, Rabbit IgG VECTASTAIN), avidin-biotin complex, 3-3'-diaminobenzidine, peroxidase substrate kit DAB (SK-4100), Permount mounting medium, coverglass, slide, pasteur pipette, micropipette, oscillator.
Troubleshooting
Before start
Check the availability of equipment and necessary supplies. Thaw the antibody aliquots if necessary.
Brain preparation, tissue collection, and storage
Anesthetize the rats by intraperitoneal injection of a ketamine/xylazine cocktail (0.75 ml ketamine + 0.25 ml xylazine + 5 ml sterile saline), administering a dose of 0.125 ml per 20 g of body weight.
Perform transcardial perfusion with 300 ml of fresh cold phosphate buffered saline (1x PBS, pH 7.4), followed by 300 ml of 4% paraformaldehyde.
Remove brains and postfix 4% paraformaldehyde for 48h in a Falcon tube.
Perform coronal brain slices of the region of interest at 40 µm using a vibratome (Leica, VT1000S microsystem, USA).
Collect slices sequentially in four-row wells coated with 1x PBS.
After sectioning all brains, remove 1x PBS and place sections in cryoprotectant solution [glycerol: ethylene glycol: 1x PBS, (3:3:4)] and store at -20 °C until further use.
Immunohistochemistry (GFAP or Iba1)
1d 5h 37m 30s
Wash slices in phosphate buffered saline (1x PBS, pH 7.4), 4 times for 5 minutes.
20m
Incubate slices in 1.5% hydrogen peroxide dissolved in 1x PBS for 5 minutes.
5m
Rinse slices in 1x PBS, 2 times for 5 minutes.
10m
Incubate slices in blocking solution [1x PBS + 0.3 % Triton X-100 + 3% normal rabbit serum (for GFAP) or 3% normal goat serum (for Iba1)] for 2 hours.
2h
Add primary antibody [goat anti-GFAP ab53554, abcam [1:500] (for astrocytes) or rabbit anti-Iba-1 cell signaling [1:1000] (for microglia)] in diluent antiserum [1x PBS + 0.3 Triton X-100 + 1% normal rabbit serum (for GFAP) or 1% normal goat serum (for Iba1)] and incubate for 24 hours at room temperature.
1d
Wash slices in 1x PBS, 3 times for 5 minutes.
15m
Incubate slices in secondary antibody: anti-goat [1:250] using a Goat IgG VECTASTAIN, Elite, ABC-HRP Kit, Peroxidase, PK-6105, CA, USA (for GFAP) or anti-rabbit [1:500] using a Rabbit IgG VECTASTAIN, Elite, ABC-HRP Kit, Peroxidase, PK-6101, CA, USA (for Iba1) dissolved in diluent antiserum for GFAP or Iba1, for 1 hour at room temperature.
1h
Wash slices in 1x PBS 3 times for 5 minutes.
15m
Incubate slices in avidin-biotin complex for 1 hour at room temperature.
1h
Wash slices in 1x PBS, 3 times for 5 minutes.
15m
Incubate slices with 3-3'-diaminobenzidine for 2:30 minutes (Peroxidase substrate kit DAB, SK-4100, Vector Laboratories Inc., CA, USA).
2m 30s
Wash slices in 1x PBS twice for 5 minutes and once in distilled water for 5 minutes.
15m
Mount slices on gelatinized slides and cover with Permount mounting medium and coverglass.
Immunohistochemistry (c-Fos)
1d 5h 37m 30s
Wash slices in phosphate buffered saline (1x PBS, pH 7.4), 4 times for 5 minutes.
20m
Incubate slices in 1.5% hydrogen peroxide dissolved in 1x PBS for 5 minutes.
5m
Rinse slices in 1x PBS 2 times for 5 minutes.
10m
Incubate slices in blocking solution (1x PBS + 3% normal rabbit serum + 0.3% Triton X-100) for 120 min.
2h
Add primary antibody (Abcam goat anti c-Fos polyclonal ab156802 at [1:500]) in diluent antiserum (1x PBS + 1% normal rabbit serum + 0.3% Triton X-100) and incubate for 24 hours at room temperature.
1d
Wash slices in 1x PBS, 3 times for 5 minutes.
15m
Incubate slices in secondary antibody (anti goat [1:250]) using a Goat IgG, VECTASTAIN, Elite, ABC-HRP Kit, Peroxidase, PK-6105, CA, USA for 1 hour at room temperature.
1h
Wash slices in 1x PBS, 3 times for 5 minutes.
15m
Incubate slices in avidin-biotin complex for 1 hour at room temperature.
1h
Wash slices in 1x PBS, 3 times for 5 minutes.
15m
Incubate slices with 3-3'-diaminobenzidine for 2.5 minutes (Peroxidase substrate kit DAB, SK-4100, Vector Laboratories Inc., Burlingame, CA, USA).
2m 30s
Wash slices in 1x PBS twice for 5 minutes and once in distilled water for 5 minutes.
15m
Mount slices on gelatinized slides and covered with Permount mounting medium and coverglass.
Protocol references
Galindo-Paredes, G., Flores, G., & Morales-Medina, J. C. (2023). Olfactory bulbectomy induces nociceptive alterations associated with gliosis in male rats. IBRO neuroscience reports, 14, 494–506. https://doi.org/10.1016/j.ibneur.2023.05.006
Monfil, T., Vázquez Roque, R. A., Camacho-Abrego, I., Tendilla-Beltran, H., Iannitti, T., Meneses-Morales, I., Aguilar-Alonso, P., Flores, G., & Morales-Medina, J. C. (2018). Hyper-response to Novelty Increases c-Fos Expression in the Hippocampus and Prefrontal Cortex in a Rat Model of Schizophrenia. Neurochemical research, 43(2), 441–448. https://doi.org/10.1007/s11064-017-2439-x