Affinipure goat anti rabbit igg h l antibody

SC protein extracts were prepared by homogenization of mouse SC in extraction buffer [25 mM Tris-HCl pH 7.6, 300 mM NaCl, 0.5% Nonidet P-40, 2 mM EDTA, 2 mM MgCl₂, 0.5 M urea and protease inhibitors (Sigma-Aldrich, P8340)] followed by centrifugation at 4°C for 20 min at 14 000 RPM. Supernatants only were used for western blotting. Protein extracts were resolved by SDS–PAGE and transferred to Hybond (Amersham) followed by detection with ECL reagent (Amersham). Antibodies included IFIH1 rabbit polyclonal antibody (1:3000, Proteintech Cat# 21775–1-AP), TRIM30 antibody (1:3000, Novus Biologicals, NBP2-41087), Anti-PCP4 antibody (1:5000, Abcam, ab197377), SQSTM1/p62 antibody (1:4000, Cell Signaling, Cat# 5114), mTOR antibody (1:4000, Cell Signaling, Cat# 2972), LC3B antibody (1:7000, Novus Biologicals, NB100-2220), GLT-1/SLC1A2/EAAT2 antibody (9 HCLC), ABfinity™ Rabbit Oligoclonal (1:3000, Thermo Fisher Scientific, Cat#: 711020) and monoclonal anti-β-Actin−peroxidase antibody, clone AC-15 (1:20 000, Sigma-Aldrich, A3854). The secondary antibody was peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H + L) antibody (1:5000) (Jackson ImmunoResearch Laboratories, Cat# 111–035-144).

Multiple immunofluorescence staining of FFPE tissue sections was carried out as described previously [11] . Antigen retrieval was performed using the same method as described above in the IHC section. A cocktail of primary antibodies was applied to the tissue sections, which were incubated at room temperature overnight. A fluorescein-conjugated AffiniPure goat anti-rabbit IgG (H+L) antibody (Jackson ImmunoResearch Laboratories; West Grove, PA, USA) and a rhodamine-conjugated AffiniPure donkey anti-mouse IgG (H+L) antibody (Jackson ImmunoResearch Laboratories) were used as secondary antibodies, and the samples were mounted using Fluoromount (Diagnostic Biosystems Inc.; Pleasanton, CA, USA). Staining was observed using a fluorescent microscope.

H. pylori from 22- to 24-h blood agar plate cultures was harvested in 1 ml cold phosphate-buffered saline (PBS) (600 × g, 4°C, 5 min). Cell pellets were suspended in 200 μl of homogenization buffer (Tris-HCl, 100 mM, pH 7.4). Samples were sonicated for 1 min at continuous pulse. Total protein concentration was calculated using the Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Darmstadt, Germany). The same amounts of protein samples were separated on SDS-PAGE gels (14%). Subsequently, Western blotting (WB) was performed for the detection of target proteins. Detection of BabA was achieved with a rabbit antiserum kindly provided by Thomas Borén (Umeå University, Sweden) and a secondary peroxidase-labeled Affini-pure goat anti-rabbit IgG (H+L) antibody (Jackson Immuno Research Laboratories, USA). Catalase detection was achieved using an antibody from the Ridascreen FemtoLab stool antigen test (R-Biopharm AG, Darmstadt, Germany) as described previously (8 (link)).
SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific, Darmstadt, Germany) was used for WB developing. The Las-3000 imaging system (Fujifilm Life Science, Düsseldorf, Germany) and chemiluminescence were used to reveal the WB.