IL-1α Protein Immunolocalization Protocol

The distribution of IL-1α protein was assessed according to previously published protocols [31 (link)]. The cells were washed in PBS, fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, incubated with 5% BSA, and then incubated with primary anti-IL-1α polyclonal antibody (1 : 50) at 4°C overnight. After overnight incubation, the cells were washed in PBS twice and incubated with secondary Alexa Fluor 488-conjugated donkey anti-rabbit IgG antibody for 2 hours (BioLegend, San Diego, CA, USA). After immunostaining, the cells were counterstained with the endoplasmic reticulum ER-ID® Red assay kit (Enzo Life Sciences, Farmingdale, NY, USA). Fluorescence was visualized using a Leica DMi8 fluorescence microscope at 40x magnification equipped with filters (A for Hoechst, GFP-EN for Alexa Fluor 488, and N21 for ER Texas Red) and analyzed by LAS EZ software.