Morphological Analysis

（1）Bake slides with 60℃ for 2h on the slide warmer;

（2）Dewaxing: xylene I, 5min → xylene I, 5min → xylene I, 5min；

（3）Hydration: 100% ethanol, 3min →  85% ethanol, 3min → 75% ethanol, 3min → PBS, 3min for three times；

（4）Antigen retrieval (microwave): citrate (high fire, 4min → middle fire, 4min → high fire, 4min → deforst, 10min);

（5）Cool down ar RT, about 2h;

（6）Wash slides with PBS for three times, each for 5min;

（7）Surround each tissue section with a hydropphobic barrier using a marking pen (optional);

（8）Blocking: 5% goat serum (from the species the secondary antibody was raised in), 200ul, cover the tissue and was incubated in a humidified sealed chamber,  1h →  don't wash!! (important!!)

（9）Primary antibody incubation(MCT-1): 50ul, cover the tissue, 4℃，overnight;

（10）Rewarming at RT for 30min;

（11）Wash slides with PBS for three times, each for 5min;

（12）Secondary antibody incubation(A): 50ul, cover the tissue, RT，2h;

（13）Wash slides with PBS for three times, each for 5min;

（14）Primary antibody incubation(MCT-4): 50ul, cover the tissue, 4℃，overnight;

（15）Rewarming at RT for 30min;

（16）Wash slides with PBS for three times, each for 5min;

（17）Secondary antibody incubation(different from A): 50ul, cover the tissue, RT，2h;

（18）Wash slides with PBS for three times, each for 5min;

（19）DAPI, 200ul, RT, 15min;

（20）Wash slides with PBS for three times, each for 5min;

（21）Protect tissue by using the anti-IF quench sealing solution with a coverslip.