Meyer s hematoxylin solution

Luteolin, Wedelolactone, DAPI, Hoechst 33342, JC-1, Doxorubicin, poly-L-lysine solution, Meyer’s Hematoxylin solution, DPX mountant for histology and β-actin were obtained from Sigma Aldrich. Control peptide, pan-caspase inhibitor and Caspase-9 inhibitor were purchased from Calbiochem. Dharma-FECT transfection reagent and Hsp60 siRNA were purchased from Dharmacon. Fluorochrome conjugated secondary antibodies, Annexin-V Alexa Fluor 488 and ER-Tracker™ Blue-White DPX were procured from Molecular Probes-Invitrogen. ImmEdge pen (hydrophobic barrier pen), Bloxall blocking solution, DAB peroxidase substrate kit, Vectastain ABC kit were purchased from Vector Laboratories, Inc. Burlingame. Anti-human mouse Hsp60, GAPDH, and β-tubulin antibodies were purchased from Thermo-Fisher whereas, anti-human rabbit Hsp60, Cleaved PARP, Caspase-8, Caspase-9, COX-IV, PDI and Calnexin were obtained from Cell Signaling Technology, Inc. XIAP, PCNA, and HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology. All chemicals and antibodies were obtained from Sigma unless specified otherwise.

Frozen secions were immersed in PBS, while paraffin sections were dewaxed, rehydrated, then immersed in PBS. Hematoxylin and eosin (H&E) staining was performed using Meyer’s hematoxylin solution (Muto Pure Chemicals, Tokyo, Japan) and 1% eosin in water (Fujifilm-Wako, Osaka, Japan). For Alcian blue staining, sections were rehydrated, stained with 1% Alcian blue 8GX (Sigma-Aldrich-Japan, Tokyo, Japan) in 3% acetic acid for 15 min, and stained with Meyer’s hematoxylin solution for 1 min, and 0.1% eosin in DW for 1 min. Microscopic images were obtained using a Nikon AZ-100 (Tokyo, Japan) and a Sony a7RII (Tokyo, Japan).

Dimethyldioctadecylammonium was obtained from Avanti Polar Lipids (Alabaster, AL, United States). TDB was purchased from Niels Clauson-Kaas A/S (Farum, Denmark). Xenolight 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR) near infra-red fluorescent dye was purchased from Perkin Elmer (Waltham, MA, United States). H56 protein was produced recombinantly in E. coli as previously described (15 (link)), reconstituted in 20 mM glycine buffer (pH 8.8), checked for purity, and validated for residual DNA, endotoxins and bioburden following internal good manufacturing practice standards at Statens Serum Institut as described previously (16 (link)). Alexa Fluor^® 647-labeling of H56 was performed commercially (Thermo Fischer Scientific, Eugene, OR, United States). 2,5-dihydroxybenzoic (DHB), 1,5-diaminonaphthalene (DAN), trifluoroacetic acid (TFA), and Meyer’s hematoxylin solution and eosin (H&E) solution were purchased from Sigma-Aldrich (St. Louis, MO, United States). Methanol was obtained from Th. Geyer (Renningen, Germany). Water was prepared by using a Millipore Direct-Q3 UV system (Billerica, MA, United States). All other chemicals and reagents were of analytical grade and were acquired from commercial suppliers.