Immunofluorescence
SECONDARY
Sections of 4 μm were dewaxed in two changes of xylene for 5 min each and rehydrated to water through graded ethanols (2 × 100%, 1 × 80%, 1 × 50%) for 5 min each. Heat‐mediated antigen retrieval was performed by incubating sections in a citrate buffer at 98°C for 20 min with the use of a water bath. After cooling to room temperature, sections were washed in several changes of 1xPBS and blocked with 5% normal goat serum for 1 h at room temperature. Following that, sections were immunostained with a rabbit polyclonal anti‐HSPB5 or rabbit polyclonal anti‐HSP25 (rodent HSPB1) antibody (both Enzo Life Sciences, NY, USA) diluted 1:250 in PBS with 1% BSA (immunohistochemical grade; Vector Laboratories) at 4°C overnight. Anti‐rabbit conjugated to Abberior STAR 580 (Abberior GmbH) was used to visualize HSPB5 and HSP25. The membranes were stained with Wheat‐Germ Agglutination (WGA) conjugated with Alexa 488 (Thermofisher) diluted 1:100 in PBS, and the nuclei were stained with DAPI (Thermofisher) diluted 1:500 in PBS. The secondary antibody, WGA and DAPI were incubated for 2 h at room temperature. The samples were washed 3x with PBS and mounted in Mowiol mounting medium +2.5% v/v DABCO (Thermofisher). The staining was visualized with a Leica TCS SP8 3X (Leica Microsystems). DAPI, Alexa 488, and Abberior 580 were irradiated with a laser at 405, 499, and 587 nm, respectively. An oil‐immersed 63x objective with a Numerical Aperture of 1.4 was used to image each section. The fluorescent signal was detected by the use of gated Hybrid Detectors. The images were analyzed, and background levels adjusted based on a negative control without primary antibody. The analyses were done with FIJI (Schindelin et al., 