Immunohistochemistry

For immunohistochemical staining on cryosections, sections were washed with 0.5% Tween20/Tris-buffered saline (TTBS), pH 7.4, and then blocked for 1 h at room temperature in 5% normal donkey serum (Jackson ImmunoResearch, West Grove, PA, USA)/0.5% TTBS. For anti-HSP70 immunostaining, antigen retrieval was performed prior to these steps by boiling in 10 mM citric acid, pH 6, for 15 min at 20% microwave power. Antibody incubations were performed overnight at 4 °C in block solution. A complete list of primary antibodies is provided in Supplementary Table 1. Sections were washed in 0.5% TTBS at room temperature prior to secondary antibody incubation overnight at 4 °C in the dark. Alexafluor-conjugated secondary antibodies included 488, 594, and 647 (Thermo Fisher Sci, Waltham, MA, USA) and were diluted at 1:500 each. 4′6-diamidino-2-phenylindole (DAPI) diluted at 1:10,000 was added during the secondary antibody incubation. Sections were washed in 0.5% TTBS and mounted in ProLong Gold Antifade (Thermo Fisher Scientific) on frosted plus microscope slides (Thermo Fisher Scientific).

For whole-mount preparations, tissues were collected in 300 μL dPBS in Eppendorf tubes, flash-frozen with liquid nitrogen, thawed on ice, and transferred to a 40-well plate. Tissues were washed in 0.5% TTBS, blocked for 1 h at room temperature in 5% normal donkey serum/0.5% TTBS, and incubated in 200 μL of primary antibody overnight at 4 °C. Tissues were washed in 0.5% TTBS at room temperature and incubated in 200 μL of secondary antibody solution overnight in the dark at 4 °C. Tissues were washed with 0.5% TTBS prior to mounting in ProLong Gold Antifade between two 50 mm coverslips (Thermo Fisher Scientific).