HCN4 immunostaining in human (Alomone labs antibody)

Preparation of human fixed brain sections:  

Frozen (unfixed) OCT-embedded tissue were cut on cryostat (30 μm thick) and mounted onto glass slides. 

Procedure: 

1. Fix sections in 4% PFA for 5 min, followed by 3x rinses in PBS. 
2. Permeabilize in PBS+ 0.3%Tx for 20 min. 
3. Block (10% goat serum, 0.3% BSA, 0.3% Tx in PBS) for 2 hours at RT. 
4. Incubate in 1°antibodies (diluted in 5% goat serum, 0.3% BSA, 0.1% Tx in PBS) o/n at RT (in humidity chamber).   
5. Wash in PBS+0.1%Tx for 3x10 min. 
6. Incubate in 2°antibodies for 2 hours at RT.  
7. Incubate in DAPI for 5 min. 
8. Wash in PBS+0.1%Tx for 3x30 min. 
9. Rinse in PBS, airdry, and coverslip with mounting media. 

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