Day 1: Whole mouse brain (2 years old, male) was fixed overnight in 4%PFA.

Day 2: Brain was washed 3x 5' in PBS + 4% Sucrose

Day2: Brain was incubated overnight in PBS + 30% Sucrose

Day3: Brain was embedded into optimal cutting medium (OCT)

Day3: Brain was cut with a cryostat in 10um thick sections and stored at -80C

Slides with brain tissue sections were thawed 0h 15m 0s Room temperature

Add slides to glass coplin Jar.

Wash 3x 0h 10m 0s in PEMTx

Move slides to plastic coplin jar for antigen retrieval

Perform antigen retrieval 0h 15m 0s 85°C

Let plastic coplin jar cool down to room temperature 0h 20m 0s

Place slides back in original glass complin jar

Wash 2x 0h 10m 0s in PEMTx

Place slides horizontally in box humidified with MQ. Use hydrophobic pen to outline tissue sections.

Add blocking buffer to the slides and incubate 1h 0m 0s

Add antibody solution (blocking buffer + 1:100 LBP110 + 1:100 p62) and incubate 4°C

Wash slides 4x 0h 15m 0s

Incubate with secondary antibody solution in humidified box (PEMTx + 2% BSA + 1:500 secondary antibodies + 1:1000 DAPI) for 1h 0m 0s

Place slides back in coplin jar (keep in dark)

Wash 3x 0h 10m 0s in PEMTx

Wash 2x 0h 7m 0s in PEM

Mount slides using Prolong Gold mounting medium and store at 4°C