Anti mouse igg conjugated to horseradish peroxidase

Protein extraction from formalin-fixed paraffin-embedded tissues was performed according to Guo and co-workers [47 (link)]. Briefly, paraffin blocks were cut (approximately 10 µm), deparaffined in xylene and rehydrated in decrescent graded alcohol (100, 90, 70, 50, 30% v/v). Samples were resuspended in 20 mM Tris pH 9 and 2% SDS and boiled for 20 min at 100 °C and 2 h at 80 °C. After centrifugation, supernatants were collected, and protein content quantified by Quick Start Bradford (Bio-Rad, Hercules, CA, USA). Fifty μg of proteins from each sample were diluted in Laemmli buffer and boiled for 5 min. Samples were subsequently resolved on SDS-PAGE and transferred to PVDF membranes. Membranes were blocked for 1 h at RT with 5% non-fat dry milk in TBS buffer (20 mM Tris and 105 mM NaCl) containing 0.1% Tween-20 (block solution) and then incubated overnight with primary antibody dissolved in block solution. The day after, membranes were washed three times in TBS containing 0.1% Tween-20 (TBST) and incubated in secondary anti-mouse IgG conjugated to horseradish peroxidase (Bio-Rad, Hercules, CA, USA) diluted 1:10,000 in block solution. Immunostained bands were detected by a chemiluminescent method (Clarity Western ECL Substrate, #1705061, Bio--ad, Hercules, CA, USA). Primary antibodies (α-SMA and tubulin) were diluted 1:1000 in block solution.

Total protein extracts of wild-type or mutant BSF of T. brucei (5 x10⁶ cells) were size-fractionated by SDS-PAGE (10%) and immunoblotted on Immobilon-P filters (Millipore) [80 ]. Immunodetection was performed as described [81 ,82 (link)] using primary antibodies, rat anti-T. brucei PEPCK (diluted 1:1000; gift from T. Seebeck, Bern, Switzerland), rabbit anti- T. brucei enolase (diluted 1:1,000, gift from P. Michels, Edinburgh, UK) and mouse anti-HSP60 (diluted 1:10 000) [83 (link)], and as secondary antibodies, anti-rat, anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase (BioRad, 1:5,000 dilution). Revelation was performed using the SuperSignal West Pico Chemiluminescent Substrate as described by the manufacturer (Thermo Fisher Scientific). Alternatively, for quantitative analyses, revelation was performed using the Luminata Crescendo Western HRP Substrate (Millipore). Images were acquired and analyzed with a KODAK Image Station 4000 MM and quantitative analyses were performed with the KODAK MI application.

Astrocytes were cultured as adherent monolayers in 75 cm² flasks at a density of 8×10⁶ cells/flask and allowed to recover for 24 h. Following recovery, cells were treated for various time points, and whole-cell protein extracts were isolated using mammalian protein extraction buffer (Thermo Fisher Scientific). Cells were collected by scraping in sterile ice-cold PBS to avoid alteration of protein expression on surface of cell membranes. Whole-cell protein extracts (25 μg) were boiled with 4× NuPAGE loading sample buffer at 100 °C for 5 min, resolved by Bolt 4–12% bis tris gel and subsequently transferred to nitrocellulose membranes using i-Blot (Thermo Fisher Scientific). The membranes were incubated with antibodies against ATF4 (1 : 1000, Cell Signaling), ATF6 (1 : 1000, Cell Signaling), XBP-1s (1 : 1000, Cell Signaling), p-eIF2α, and eIF2α (1 : 1000, Cell Signaling) overnight at 4 °C, washed, and then incubated with anti-rabbit IgG conjugated to horseradish peroxidase (1 : 10 000, Bio-Rad, Hercules, CA, USA) or anti-mouse IgG conjugated to horseradish peroxidase (1 : 10 000, Bio-Rad) for 2 h at room temperature. The membranes were then developed with SuperSignal west femto substrate (Thermo Fisher Scientific) and imaged using Fluorochem HD2 imager (ProteinSimple, San Jose, CA, USA). GAPDH (1 : 2000, Santa Cruz Biotechnology, Dallas, TX, USA) was used as a loading control.