Horseradish peroxidase hrp conjugated goat anti rabbit secondary antibody

Part of the ventricle was homogenized in lysis buffer and protein levels were assessed using a Bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific Inc. United States). Protein expression was estimated as previously described (Ahmed et al., 2014) using troponin I primary antibody (Thermofisher scientific, United States, Cat.# MA1-20112), troponin T primary antibody (Thermofisher scientific, United States, Cat.# MA1-24611), forkhead/winged helix transcription factor P3 (FoxP3) primary antibody (Thermofisher scientific, United States, Cat.# PA5-23169), retinoic-acid-related orphan receptor (ROR)-γt primary antibody (Thermofisher scientific, United States, Cat.# PA5-23148) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Thermofisher scientific, United States, Cat.# 31460). The amount of protein was quantified by densitometric analysis of the autoradiograms using a scanning laser densitometer (Biomed Instrument Inc. United States). Results were normalized to β-actin and expressed as fold change to the normal group.

M. avium subsp. hominissuis isolates were cultured for 2 weeks in 7H9 broth. Once turbid, the cultures were centrifuged at 5,000 × g for 10 min, supernatant was removed, and pellets were stored at –80°C. Extraction of M. avium subsp. hominissuis total proteins was performed using B-PER (Thermo Fisher Scientific, Waltham, MA) supplemented with DNase I and lysozyme (Thermo Fisher Scientific). Twenty micrograms of M. avium subsp. hominissuis total proteins and Mycobacterium tuberculosis strain H37Rv culture filtrate proteins (BEI Resources NR-14825) was separated by SDS-PAGE on a 4 to 12% Bis-Tris–morpholineethanesulfonic acid (MES) gel (Thermo Fisher Scientific) and transferred onto a polyvinylidene difluoride (PVDF) membrane (iBlot, Invitrogen). Membranes were blocked in 5% nonfat milk-PBS with 0.25% Tween 20 (PBST) overnight at 4°C. Polyclonal anti-LAM primary antibody (BEI Resources NR-13821) was diluted 1:1,000 in 5% nonfat milk in PBST and used with 1:1,000 horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Thermo Fisher Scientific). NR-14825 and 13821 reagents were obtained through BEI Resources, NIAID, and the NIH. Blots were visualized using SuperSignal West Femto maximum sensitivity substrate (Thermo Fisher Scientific) on the iBright CL1000 imager (Invitrogen, Carlsbad, CA).

Capsular polysaccharide (CPS) was quantified by immunoblotting, as described [32 (link)]. An isogenic capsular variant of TIGR4 (T4R) in which the cps locus is replaced with the Janus cassette resulting in an unencapsulated phenotype [33 ] was used as a control. Briefly, bacterial strains were cultured in THY supplemented with 10% fetal bovine serum to an OD_{600 nm} of 0.2, plated on BAP for CFU enumeration and 1 mL bacteria was pelleted and stored at −20 °C until further use. The CFUs for all strains were ~ 9.0 × 10⁷/mL. CPS was extracted in a lysis buffer (4% deoxycholate, 50 μg/mL DNAseI and 50 μg/mL RNAse) at 37 °C for 10 min and centrifuged at 18,000× g for 10 min. Four threefold serial dilutions of the supernatant in phosphate-buffered saline (PBS) were spotted in duplicate (2 μL) on 0.2-μm-pore-size nitrocellulose membrane (Thermo Fisher Scientific, Waltham, MA, USA) with suction and air dried at 60 °C for 15 min. The membranes were blocked and incubated with rabbit anti-serotype 4 serum (Cedarlane, Burlington, NC, USA) at 1:1000 and a horseradish peroxidase (HRP) conjugated goat anti-rabbit secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) at 1:10,000. Membranes were developed with enhanced chemiluminiscence (ECL) detection (Thermo Fisher Scientific, Waltham, MA, USA) and scanned using a ChemiDoc XRS+ with Image Lab software (Bio-Rad, Hercules, CA, USA).

Lipopolysaccharide (LPS) was obtained from Sigma Aldrich (China). PGZ was purchased from SelleckChem (China). The assays for the detection of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and IL-10 were purchased from Beyotime Biotech (China). The synthetic short interfering RNA (siRNA) targeting PGC-1α (siPGC-1α) and its corresponding negative control (NC) were purchased from GenePharma (China). The antibodies against PPARγ, nuclear respiratory factor 1 (NRF1), NRF2, mitochondrial transcription factor A (mtTFA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody were purchased from ThermoFisher Scientific (USA).

Adult crude worm extracts and Hc-ESPs were quantified and diluted to 1 µg in 100 µl in ELISA coating buffer (sodium bicarbonate buffer, pH 9.0) and then loaded onto 96-well plates for overnight incubation at 4 °C. The plates were blocked with 0.05% Tween-20 and 1× PBS (PBS-Tween) containing 5% bovine serum albumin (BSA) and then incubated with polyclonal antibodies against rHc-CBP-1 or rHc-CBP-2 (1:200) in 1× PBS containing 5% BSA. After washing, the wells were incubated with horseradish peroxidase (HRP) conjugated goat anti-rabbit secondary antibody (1:1000) (Thermo Fisher Scientific, Carlsbad, CA) for 1 h and then washed with PBS-Tween-20. Substrate (ABTS; 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) was added, and the reaction was stopped using 1% sodium dodecyl sulfate (SDS). Plates were read at 410 nm using a SpectraMax spectrophotometer (Molecular Devices, San Jose, CA). All samples were assayed in triplicate.