Preparation of brain samples for immunofluorescence, immunoblot, and nucleic acid analysis

For combo processing of brain tissue (1/2 of brain used for imaging, 1/2 used for biochemical/nucleic acid analysis)

1. Anesthetized mice were perfused with ice-cold PBS containing heparin (1 U/ml)

2. Mouse brain was carefully harvested and the right and left hemispheres were separated via a midline cut.

3. Each hemisphere was briefly washed in ice-cold PBS.

4. 1 hemisphere was then flash frozen in liquid nitrogen and stored at -80 until processing for protein or RNA for downstream analysis as detailed below.

5. 1 hemisphere was placed in 4% PFA in PBS and allowed to incubate at 4℃ overnight and subsequently processed starting at step 3 as detailed below for immunofluorescence staining.

Processing of brain tissue for immunofluorescence

1. Anesthetized mice were perfused with ice-cold PBS containing heparin (1 U/ml)

2. Following PBS perfursion mice were perfused with 4% PFA.

3. Following dissection, brains were post-fixed in 4% PFA overnight at 4℃.

4. Fixed brains were then transfered to 30% sucrose in PBS for cryoprotection and incubated at 4℃ overnight.

5. Brains were then embedded in OCT compound and subsequently sectioned on a cryo-sliding microtome and transfered to postively charged microscope slides.

6. Slides were then either stored at -20℃ or used directly for staining.

Processing of brain tissue for protein or RNA

1. Frozen brain tissue was cut as desired for either whole brain analysis or invidual area analysis as needed.

2. For RNA isolation and processing for RT-qPCR, tissue was processed using the Qiagen RNeasy Miniprep Kit per the manufacturer's instructions.

3. For protein isolation, tissue was homogenized via dounce homogenization in RIPA buffer as described in the primary methods section of the article.

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