Protocols - Immunofluorescence: General Animal Brain Tissue
Animal surgery and tissue processing:
General staining procedure*:
- Inject colchicine into the lateral ventricle (mouse 5 ml ~ 30mg; rat 15 ml ~ 90mg).
- Transcardially perfuse animals 24 hours after surgery (4% paraformaldehyde in 0.1M Phosphate Buffer, pH 7.4).
- Dehydrate brains in 10% sucrose (>48 hours) and snap freeze.
- Cut 16mm thick sections on a cryo-microtome and thaw mount onto gelatin-coated glass slides. Store tissue sections (slides) at -20˚C until ready to use.
- Remove tissue sections (slides) from the freezer and keep at room temperature for at least 30 minutes.
- Pre-rinse tissue sections in PBS (Phosphate Buffered Saline, pH 7.4) for 30 minutes.
- Incubate the tissue section with the primary antibody (16-24 hours at 4˚C). (Other labeling antibodies can be incubated during this step as long as these are raised in different species, e.g. Mouse Anti-GFAP.)**
- Wash the tissue section twice for 30 minutes in TBST (Tris Buffered Saline, pH 7.4 with 0.05% Tween 20).
- Incubate the tissue section with the blocking buffer for 30 minutes.
- Incubate the tissue section with a peroxidase conjugated secondary antibody for 60 minutes in the blocking buffer or according to the manufacturer’s instructions.
- Incubate the tissue section with Hoechst stain for 30 minutes in wash buffer.
- Wash the tissue section once for 30 minutes.
- Incubate the tissue section with a fluorescein Tyramide Signal Amplification (TSA™) reagent for 15 minutes or according to the manufacturer’s instructions.
- Wash the tissue section twice for 30 minutes.
- Incubate the tissue section with any additional fluorescent conjugated antibodies (e.g. Mouse Anti-GFAP) or fluorescence affinity reagents according to the manufacturer’s instructions. Wash the tissue section for 30 minutes and continue with the staining protocol.
- Incubate the tissue section in 70% ethanol for 2 minutes.
- Incubate the tissue section in Sudan Black (1% Sudan Black in 70% Ethanol) for 15 minutes.
- Destain the tissue section in 70% ethanol until the desired staining is reached.
- Coverslip the tissue section with the appropriate water-based mounting media.
* Information is courtesy of the Tissue Profiling group, SciLifeLab Stockholm. All experiments were performed according to standard protocol (Mulder et al, 2007; Mulder et al, 2009): Mulder J, Bjorling E, Jonasson K, Wernerus H, Hober S, Hokfelt T, Uhlen M (2009) Tissue profiling of the mammalian central nervous system using human antibody-based proteomics. Mol Cell Proteomics. 2009 Jul;8(7):1612-22. Mulder J, Wernerus H, Shi TJ, Ponten F, Hober S, Uhlen M, Hokflet T (2007) Systematically generated antibodies against human gene products: High throughput screening on sections from the rat nervous system. Neuroscience. 2007 Jun 8;146(4):1689-703.
** For mouse primary antibodies, please see protocol step number 11.