Goat anti rabbit hrp antibody

AD293 cells were transfected with P2A or P2A T2A stall constructs and harvested 24 hours after transfection. Cells were pelleted (200 g, 6 mins) and resuspended in RIPA lysis buffer (150mM NaCl, 50 mM Tris pH 8.0, 1% v/v IGEPAL, 0.5% v/v sodium deoxycholate, 0.1% v/v SDS, 250 U Benzonase and supplemented with cOmplete, EDTA-free Protease Inhibitor Cocktail pills (Roche)) and incubated on ice for 30 minutes. Lysate was matched for total protein by BCA kit (Thermofisher scientific, cat# 23225). 10 μg of total protein lysate was loaded on to an TGX Stain Free FastCast Acrylamide gel (BioRad, cat# 1610185) and transferred using an iBlot2 gel transfer device (Thermofisher scientific, cat# IB21001) and a PVDF iBlot2 transfer stack (Thermofisher scientific, cat# IB24001). The membrane was blocked with 5% w/v skim milk powder in phosphate buffered saline (PBS) for 1 hour at room temperature. Anti-GFP (Invitrogen, cat#A6455) and anti-Cherry (Abcam, cat#167453) antibodies were diluted to 1:10,000 and 1:2500 respectively in PBS containing 0.1% v/v Tween 20 and incubated for 1 hour at room temperature. The secondary antibody, goat anti-rabbit HRP antibody (Invitrogen, cat#656120), was diluted 1:10,000 in PBS containing 0.1% v/v Tween 20 and incubated for 1 hour at room temperature. HRP was detected by enhanced chemiluminescence.

Tetramic RI peptide (MW 4793) was synthesized by Lifetein (Somerset, NJ). Conjugation of Tetramic RI peptide and Mal-PEG4 was performed by Biosynthesis (Lewisville, Texas). The conjugation was purified and assayed using LC-MS by Biosynthesis. Successful conjugation was confirmed using ELISA for detection of tetramer peptide levels in the conjugate. On a microplate, different concentrations of conjugated peptide (0, 1, 5, 10 ng) were added in triplicate and incubated for 1 h at 37°C. The microwells were then blocked with 3% gelatin in TBS for 1h at 37°C. Wells were probed with rabbit anti-RI-peptide antibody [12 ] at 1/2500 in TBST for 1h at 37°C, then washed 10 times with TBST. The wells were treated with goat anti-rabbit HRP antibody (1/5000, Invitrogen) for 1h at 37°C. After washing the wells 10 times with TBST, 100 μl HRP substrate solution (SureBlue, KPL, Gaithersburg, MD, USA) was added and incubated for 1min at RT. One hundred microliters of 1N HCl was added to stop the reaction and the absorbance was read on Elx800 plate reader at 450nm.

The plate was precoated with a specific antibody to tau (sc-32274, Santa Cruz Biotechnology Inc.) and left overnight at 4°C. BSA 1% was added and the plate was incubated for 1 h at 37°C. RBCs (0.4 mg-100 μl) and brain tissues (2 μg-100 μl) in each well were incubated at 25°C for 2 h. Samples were probed with polyclonal antibody to tau (sc-5587, Santa Cruz Biotechnology Inc.) and incubated at 37°C for 2 h. A goat anti-rabbit-HRP antibody (Invitrogen) was incubated for 1.5 h [11 (link), 32 (link), 36 (link)].

Slides were rehydrated with xylene and decreasing concentrations of ethanol in water, blocked with endogenous peroxidase with 3% H₂O₂, and boiled for antigen retrieval in citrate buffer (pH 6.0). Sections were blocked with BSA 5% normal goat serum for 1 hour followed by overnight incubation with p16^INK4a mouse anti-human antibody (Roche Diagnostic, Clone E6H4, #705-4793, ‎Rotkreuz, Switzerland). After washing in TBST buffer, sections were incubated in goat anti-mouse HRP antibody (Invitrogen, Cat #31430, Carlsbad, CA) for 30 min in blocking buffer and stained with TSA Cy5 (Akoya Biosciences, Cat #NEL745001KT, Menlo Park, CA) for 10 min. Antibodies were stripped with a second round of antigen retrieval in citrate buffer (pH 6.0) following the TSA manufacturer’s protocol. After blocking steps, slides were incubated with rabbit anti-human TMPRSS2 antibody (#ab92323, Abcam) for 12 hours, washed, and incubated with secondary goat anti-rabbit HRP antibody (#31460, Invitrogen) for 30 min followed by 10 min of TSA FITC (Akoya Biosciences Cat #NEL741001KT). Slides were mounted in Prolong Gold anti-fade with DAPI (Thermo-Fisher Cat #P36935).

BMDEo from culture day 14 were centrifuged and resuspended in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol, and 1% NP-40) with Roche cOmplete Protease Inhibitor Cocktail and incubated at 4°C for 20 min. Nucleic acids were digested by addition of benzonase (#E1014; Sigma-Aldrich) and further incubation for 10 min at 37°C. After centrifugation at 17,000 g for 10 min at 4°C, the supernatant was transferred to a new tube, and the protein concentration was determined using Bradford assay. Samples were adjusted to a protein concentration of 1 µg/μl in Laemmli buffer with 10% β-mercaptoethanol and denatured for 5 min at 95°C. 15 µg protein was used per sample and resolved on 4–15% Mini-PROTEAN TGX Precast Protein Gels (#4561085; Bio-Rad) and transferred on a Trans-Blot Turbo Midi PVDF membrane (Bio-Rad) via semidry transfer. Membranes were washed, blocked in 1% skim milk, and incubated with polyclonal rabbit anti-AHR (#BML-SA21; Enzo), 1:2,000 in blocking buffer overnight at 4°C. The membrane was washed in Tris-buffered saline with 0.1% Tween 20, incubated with goat anti-rabbit HRP antibody (Thermo Fisher Scientific), and developed using ECL Plus reagent (Thermo Fisher Scientific). As a loading control, membranes were incubated with anti-GAPDH (#2118; Cell Signal) and developed as above with ECL reagent onto a photo film.

High-binding half area plates (Costar, Sigma-Aldrich) were coated overnight with Syn-1 (Clone 42, BD Biosciences, San Jose, CA) as capturing antibody (50 ng/well, 610,787, BD Biosciences), diluted in PBS. Blocking with 1% bovine serum albumin was incubated on shaking for 2–4 h. Samples were added to wells and incubated at RT, shaking for 2 h, or at 4 °C O/N. The IC fractions were diluted 1000–2000 times and the FFP fractions 20–200 times. The EV fractions were not diluted prior to ELISA. Alpha-synuclein monomer standards (1.95–125 pM) were diluted in either RIPA lysis buffer or in regular ELISA incubation buffer. The polyclonal FL-140 (50 ng/well, sc-10,717, Santa Cruz Biotechnology) was used as primary detection antibody. For secondary detection, a goat anti-rabbit HRP antibody (1:5000, 31,460, Thermo Fisher Scientific) was applied followed by the K-blue aqueous substrate (TMB). Finally, 1 M H₂SO₄ stop solution was added and absorbance was read at 450 nm (Infinite M1000, Tecan, Männedorf, Switzerland). Three independent experiments were analyzed and samples were run in duplicates.

MCF-7 and ZR-75-1 BC cells (ATCC) were maintained in RPMI-1640 media (Gibco, Invitrogen, USA); NPA and ARO (gifted by Mr. A. Chakraborty, BARC, India) were maintained in Iscove's modified dulbeccos medium (IMDM) (Gibco, Invitrogen, USA) containing 10% fetal bovine serum (FBS) and 0.075% gentamycin solution. HDACi MS-275 (1590-1), chidamide (2261), AR-42(2716-1), and CI-994 (1742-10) NaB and VPA were procured from Biovision. EXPOSE horseradish peroxidase (HRP)/3,3'-diaminobenzidine (DAB) detection IHC kit was from Abcam (ab80436), and MTT (3-[4, 5-dimethylthiazol-2-yl] c-2, 5-diphenyltetrazolium bromide) was from Sigma (USA). D-luciferin was procured from Biosynth Chemistry and Biology (L-8220), and luciferase assay system was from Promega (E4030). TF activation and promoter binding array (FA2002) was from Signosis (USA). FOXA1 primary antibody was from Abcam (ab-23738), H3 acetylation antibody (06-598) from Upstate (USA), human sodium iodide symporter antibody from Thermo Scientific (MA5-12308), alpha tubulin primary antibody from Sigma (T9026), γ H2Ax from Pierce Biotechnology (USA), anti-mouse HRP secondary antibody from Abcam (ab6728), goat anti-rabbit HRP antibody from Thermo Scientific (31460), and goat anti-mouse DyLight 633 secondary antibody from Thermo Scientific (35512). cDNA first strand synthesis kit was from Invitrogen (USA).