Hrp conjugated anti rabbit secondary antibody

Growth conditioned cell culture supernatants with and without 25 mM DTT were analyzed for identity and molecular mass on 8–12% NuPage SDS-PAGE gel in MES running buffer and transferred to a PDVF membrane using standard protocol for iBlot (ThermoFisher Scientific, Waltham, MA). Membrane was blocked in 5% milk overnight on shaker at room temperature then washed 3 times with phosphate buffer saline with 0.01% Tween 20 (PBST; Sigma-Aldrich, St. Louis, MO) for 10 min. Membrane was probed with 5 μg/ml rabbit polyclonal anti-rgp120 antibody from previous immunization study (PB94) (71 (link)) in 5% milk for 2 h on shaker at room temperature, washed, then probed with 1:5,000 dilution of HRP-conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA) in 5% milk for 2 h on shaker at room temperature and washed again. Antibody was detected using WesternBright reagents (Advansta, Menlo Park, CA) and visualized using an Innotech FluoChem2 system (Genetic Technologies Grover, MO).

Contralateral VB from mice with the adeno-associated virus (AAV) vector expression (see below) or from rats 1 or 7 days after CFA injection were collected and immediately stored at −80 °C. Membrane proteins were extracted using the Membrane and Cytosol Protein Extraction Kit (Applygen Technologies Inc., Beijing). Equal amounts (15 μg) of membrane proteins of gamma2/delta subunits and total proteins of GAD65 were separated by SDS–PAGE on 10% gels and transferred to PVDF membranes (Merck Millipore). Membranes were blocked with 5% milk for 30 min at the room temperature and incubated at 4 °C overnight with rabbit anti-GABA_AR primary antibodies (for the gamma2 subunit, 1:1000, Novus; for the delta subunit, 1:1000, Merck Millipore) and anti-GAD65 primary antibodies (1:5000, Abcam). The blots were then incubated with HRP-conjugated anti-rabbit secondary antibody (1:2000, Jackson, Bar Harbor, Maine). The bands were detected using an enhanced chemiluminescence detection kit (ECL, Santa Cruz Biotechnology, Inc.). Band intensity was quantified using the software Quantity One 4.6.2 (Bio-Rad Laboratories).

N2A cells were co-transfected with pcDNA3-Htt(Q)₂₅-eYFP or pcDNA3-Htt(Q)₁₀₉-eYFP and either pcDNA3 vector or pcDNA3.1-FKBP12–V5. After 48 hours, the transfected cells were harvested in 500 μl of ice-cooled PBS buffer containing protease inhibitor cocktail (Roche) and Benzonase Nuclease (Merck Millipore), and sonicated on ice for 1 min. Extracts were centrifuged at 14 K rpm for 30 min at 4 °C, and concentrations were determined using the BCA assay. Samples containing 120 μg of total proteins in a volume of 500 μl were filtered with a 0.22 μm filter (Millipore) and fractionated with a Superdex 200 10/300 column (GE Healthcare) using a flow rate of 0.3 ml/min. Each fraction (1 ml volume/fraction) was collected and subjected to Western blot and slot blot analysis. Htt oligomeric species in each fraction were quantified by densitometry (Image J). The percentage of Htt species was calculated by dividing the density of each fraction by the summed density of every fraction (fractions 7–18). For slot-blotting analysis, the collected fractions were applied to a 0.45 μm nitrocellulose membrane (Schleicher & Schuell) and probed with an A11 antibody (1:1000, Invitrogen) overnight at 4 °C, followed by incubation with HRP-conjugated anti-rabbit secondary antibody (1:7500; Jackson ImmunoResearch) for 1 hour at room temperature. Blots were detected using an ECL chemiluminescent kit.

Pancreas and liver were harvested and fixed in 4% paraformaldehyde overnight, followed by 30% sucrose incubation. Then, samples were OCT embedded, frozen, and microtome sectioned at 6 μm. The procedures of immunostaining were performed as described before^{19 (link),29 (link),30 (link)}. Secondary antibodies for fluorescent staining were Cy2-, Cy3-, or Cy5-conjugated anti-guinea pig, or anti-rabbit (Jackson ImmunoResearch, West Grove, PA, USA). Nuclei were stained with DAPI. Mouse islets were isolated by thermolysin and liberase (Roche Diagnostics, Basel, Switzerland) perfusion into the common bile duct, pancreas digestion, islet purification, and islet picking. Total proteins were extracted from isolated islets, and quantified by bicinchoninic acid method (BCA kit, Thermo Scientific, Waltham, MA, USA). SDS-PAGE was also performed as described previously^{29 (link)}. Proteins were transferred to PVDF membrane, and blotted with primary antibodies, and then identified by reacting the membrane with HRP-conjugated anti-rabbit secondary antibody (Jackson Labs, Bar Harbor, ME, USA), followed by enhanced chemiluminescence (ECL).

JIMT-1 and DU4475 cell lysates were separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). Immunoblots were probed with a rat anti-CD276 monoclonal primary antibody (clone M3.2D7, ThermoScientific, #16-5973-81), followed by HRP-conjugated anti-rat secondary antibody (Jackson Immunoresearch Laboratories, #112-035-143). For HER2 detection, lysates separated by SDS-PAGE were transferred to PVDF and probed with rabbit anti- HER2/ErbB2 antibody (M45) (Cell Signaling, #3250S), followed by HRP-conjugated anti-rabbit secondary antibody (Jackson Immunoresearch, #111-035-003). CD276 and HER2 were visualized using Pierce SuperSignal West Dura Substrate (ThermoFisher Scientific, #34076) according to the supplier’s instructions, and developed using a GeneGnome ECL processor (Syngene).

CD300f expressing I.29 cells were activated with 100 ng/ml IL-5 (Peprotech, Rehovot, IL) for 0–10 minutes. Ice-cold lysis buffer containing 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol (Pierce, Thermo Fisher Scientific, Waltham, MA) was then added to the cells. Cell lysates were directly mixed in SDS-PAGE loading buffer, resolved by SDS-PAGE, and transferred onto PVDF membrane (Millipore). Membranes were blocked with 5% skim milk in TBST (20 mM Tris pH 7.4, 150 mM NaCl, 0.05% Tween-20) for 1 h and then probed with anti-phospho-AKT, anti-AKT (Cell Signaling, Danvers, MA) overnight, followed by HRP-conjugated anti-rabbit secondary antibody or HRP-conjugated anti-Armenian hamster antibody (Jackson ImmunoResearch, West Grove, PA) for 1 h. Antibodies were diluted in either 2.5% skim milk in or 5% BSA in TBST according to manufacturer’s instructions. Blots were developed with Luminata Crescendo Western HRP Substrate and visualized on using the fusion pulse 6 chemiluminescent instrument (Analis, Belgium).