Pe conjugated anti hsp70 clone w27

Immunofluorescent imaging was performed as previously described [99 (link)]. Briefly, tumor-bearing and contralateral kidneys were harvested on day 8 of the tumor challenge/therapy scheme described above (1 day post 100μg MD5-1 mAb treatment). Tissues were snap frozen in OCT, cut to 7 μm thickness, and fixed in acetone for 10 min at −20°C. To assess tumor cell death in vivo, sections were stained using unconjugated rabbit anti-cytokeratin 8 (NB100-91850) and 18 (NBP1-67610; Novus Biologics; Littleton, CO), AF488-conjugated donkey anti-rabbit secondary Ab (Jackson ImmunoResearch Laboratories; West Grove, PA), PE-conjugated anti-CD31 (MEC13.3; eBioscience), and AlexaFluor 647-conjugated anti-human/mouse cleaved PARP (Asp214, clone F21-852; BD Pharmingen). To assess HSP70 expression in vivo after Minnelide treatment, sections were stained using unconjugated rabbit anti-cytokeratin 8 (NB100-91850) and 18 (NBP1-67610; Novus Biologics; Littleton, CO), AF488-conjugated donkey anti-rabbit secondary Ab (Jackson ImmunoResearch Laboratories; West Grove, PA), and PE-conjugated anti-HSP70 (clone W27; Santa Cruz Biotechnology) or mouse IgG2a isotype control (BioLegend). Cover slides were applied using ProLong Gold® Antifade Mountant with DAPI (Life Technologies) and imaged with a Leica DM5500 B microscope.

Untreated and triptolide-treated cells were blocked using a cocktail of anti-CD16/32 and normal mouse serum in PBS containing 2 mg/ml bovine serum albumin and 0.02% NaN₃ (FACS buffer) prior to surface staining with the PE-conjugated DJR1 (eBioscience), DJR2-4 (eBioscience), 90906 (R&D Systems), 104918 (R&D Systems) specific for TRAIL-R1, -R2, -R3, or -R4, respectively, or a PE-conjugated mouse IgG1 isotype control mAb (eBioscience) at 4°C for 30 min. Cells were analyzed immediately or fixed in 2% paraformaldehyde for 30 min at 4°C, washed with FACS buffer, and stored until analysis. For intracellular staining of HSP70, cells were permeablized in FACS buffer containing 0.5% saponin at 4°C for 30 min, and incubated with PE-conjugated anti-HSP70 (clone W27; Santa Cruz Biotechnology, Dallas, TX) or mouse IgG2a isotype control (BioLegend, San Diego, CA) at 4°C for 30 min. After staining, cells were analyzed on a BD LSR II and FlowJo software.