Anti calreticulin antibody

Hypericin was prepared/purified/stored as described in the past [64 (link)]. Tauroursodeoxycholic acid (TUDCA) was purchased from Merck-Millipore (Darmstadt, Germany) while Z-Val-Ala-Asp(OMe)-fmk (zVAD-fmk) was purchased from Bachem (Weil am Rhein, Germany). Mitoxantrone (MTX) and Anti-actin antibody was purchased from Sigma (St. Louis, MO, USA). Topotecan and paclitaxel were kind gifts from Prof. Johan Swinnen/Niamat Ali Khan (KULeuven, Belgium). Antibodies against CHOP/P-eIF2α/Total eIF2α/cleaved caspase 3/PARP/HSP90/Bip/GRP78 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-calreticulin antibody (for immunoblotting) was purchased from Stressgen (Victoria, BC, Canada). Antibody against HMGB1 was purchased from Abcam (Cambridge, UK). Anti-HSP70 antibody was purchased from Santa Cruz Antibodies (San Francisco, USA). Secondary antibodies conjugated to horseradish peroxidase were purchased from Cell Signaling Technology (Danvers, MA, USA)/Abcam (Cambridge, UK). Also, the following secondary antibodies were used: goat anti-mouse-DyLight680/goat anti-rabbit-DyLight800 (Thermo Scientific, Belgium).

For immunolabeling, ultrathin sections (60 nm thickness) were washed three times with PBS and three times with PBS containing 1 % BSA and 0.15 % glycine, followed by a 30-min blocking step with 5 % normal goat serum. Samples were incubated with the anti-TG2 (CUB 7402) diluted 1:25 for 1 h at room temperature. Where indicated, an anti-calreticulin antibody (Stressgen) was used. After being washed in PBS, samples were incubated with appropriate secondary antibody conjugated to 15-nm or 5-nm gold particles (BioCell, Cardiff, UK). Sections were stained with 2 % uranyl acetate and observed under a Zeiss EM900 transmission electron microscope. Images were captured digitally with a Mega View II digital camera (SIS, Soft Imaging System, Münster, Germany).