Tissue section preparation 1. Anesthetize mice with an overdose of pentobarbital and perfuse intracardially with PBS, followed by paraformaldehyde (PFA, 4% wt/vol in PBS). 2. Dissect tissues and postfix them in 4% PFA for 4 h at room temperature or 1 day at 4°C. 3. Dehydrate tissue samples in 30% sucrose solution. 4. Prepare thin tissue sections on a Cryostat microtome. Immunostaining 1. Wash and permeabilize sections in 0.3% Triton X-100 in PBS (PBST) and block them in 2% BSA in PBST at room temperature for 1 h. 2. Incubate sections with primary antibodies for various times according to the efficiency of the antibodies. 3. Wash samples three times in PBST. If DNA HCR initiator conjugated secondary antibodies are used, incubate samples in HCR amplification buffer [5× sodium chloride citrate (SCC buffer), 0.1% vol/vol Tween-20, and 10% wt/vol dextran sulfate in ddH2O] for 30 min at room temperature. 4. Incubate sections with biotinylated secondary antibodies or DNA HCR initiator conjugated secondary antibodies. 5. Wash samples three times in PBST. isHCR amplification 1. Snap-cool a pair of DNA-fluorophore HCR amplifiers separately in 5× SSC buffer by heating at 95°C for 90s and cooling to room temperature over 30 min. 2. Both amplifiers are then added to amplification buffer to a final concentration of 12.5 nM. Optional: Graphene oxide (GO) can be added for applications that demand background suppression. Add GO to the buffer from step 2 to 20 µg/mL and vortex thoroughly. 3. Incubate samples with the buffer from step 2 overnight at room temperature. 4. Wash samples three times in PBST. 5. Mount sections on slides and observe.