Anti mouse igg hrp

The slices were baked at 60°C for 12 h. The slices were dewaxed and heated to repair the antigen. An appropriately diluted primary antibody GFAP (556330, BD Pharmingen) was added overnight at 4°C. 50–100 μL anti-mouse IgG-HRP (Bio-Rad, Hercules, CA) was added and incubated at 37°C for 30 min and then washed with PBS for 5 min 3 times. Diaminobenzidine (DAB, ZSGB-BIO, China) was used to be a chromogen. The samples were placed in xylene for 10 min 2 times. The neutral gum was sealed and enforced under a microscope.

The reactivity of the MAbs to PLY and related cytolysins was evaluated by WB. Recombinant cytolysins at 1 µg/lane or 20 µL of bacterial lysate after boiling in a reducing sample buffer (39000, Thermo Fisher Scientific) were electrophoretically fractionated in 12.5% SDS-PAGE mini gel using SDS-Tris-Glycine buffer. Proteins from the SDS-PAGE were transferred to the polyvinylidene-difluoride (PVDF) membrane (T830.1, Carl Roth, Karlsruhe, Germany) under semidry conditions. The membranes were blocked with 2% milk powder (T145.3, Carl Roth) in PBS by incubating overnight at 4 °C. After blocking, the membranes were incubated for 1 h at RT with the purified MAbs (for 1 cm² membrane sheet, 0.2 mL of the MAb at a concentration of 2 µg/mL were used). After washing in PBST, the membranes were incubated for 1 h with anti-mouse IgG-HRP (1721011, Bio-Rad) at RT. After rinsing the bands of proteins were developed with ready-to-use TMB solution (37574, Thermo Fisher Scientific). The reaction was stopped by immersing the membranes into water.
The reactivity of the MAbs with the wild-type PLY from cultured clinical S. pneumoniae isolates, was performed by WB as described above. The clinical isolate cultures 6477, 6840, 6958, 7123, 7144 were obtained from The National Public Health Laboratory (Vilnius, Lithuania) as lysates in PBS with 2% of sodium dodecyl sulphate (SDS).

Protein lysates were prepared and analyzed by a conventional western blot (WB) assay as described elsewhere [39]. Signals from enhanced chemiluminescence reagent (ECL, Amersham, Piscataway, NJ, USA), used in the WB assay, were acquired by a ChemiDoc XRS + (Bio‐Rad Laboratories, Hercules, CA, USA) with image lab™ software (Bio‐Rad Laboratories, Hercules, CA, USA). Antibodies, anti‐ATGL (sc‐365278, RRID: AB_10859044; Santa Cruz Biotechnology) at 1 : 1000, actin (C4) : sc‐47778, and anti‐mouse IgG‐HRP (170‐6516), were obtained from Bio‐Rad Laboratories.

Whole cell protein lysates were prepared using RIPA buffer containing 0.2% SDS, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM PMSF and EDTA-free mini-complete protease inhibitor cocktail tablet (Roche). Tissue samples or cells were homogenized in RIPA Lysis Buffer. The protein concentration was determined using Bradford’s Reagent (BioRad). 40 µg of protein was subjected to polyacrylamide gel electrophoresis and transferred on to a nitrocellulose membrane (Merck Millipore). Membranes were incubated with respective primary antibody dilutions prepared in Tris Buffered Saline (TBS)-Tween for 2 h at room temperature or overnight at 4 °C. Membranes were then washed in TBS-Tween and incubated with secondary antibodies anti-rabbit IgG–HRP (BioRad) or anti mouse IgG-HRP (BioRad) diluted (1:10.000) in TBS-Tween for 1 h at room temperature. Protein-antibody complexes were detected by Substrate Detection Kit (Thermofischer, USA). Quantification of signal was done via densitometry analysis, using the Image J software. Actin was used as loading control for whole cell lysates and Lamin A/C and GAPDH were used as loading controls for nuclear and cytoplasmic fractions respectively.

Protein samples were denatured by boiling at 95°C for 3 min in protein-loading buffer [2% (w/v) SDS, 100 mM DTT, 0.05% (v/v) BPB, and 10% (v/v) glycerol], resolved by SDS-PAGE, and transferred to 0.2-μm polyvinylidene difluoride membrane (Wako) using the semi-dry system (Trans-blot Turbo, Bio-Rad). The membrane was blocked in 4% (w/v) skim milk (Nacalai) in 1× phosphate-buffered saline (PBS) supplemented with 0.1% (v/v) Tween 20 and further incubated with the following antibodies: anti-Aub antibody (1:500; guinea pig) (80 (link)), anti-GFP antibody (1:2000; Clontech, rabbit), anti-Ago1 antibody (1:1000; Abcam, Ab5070, rabbit), anti-Ago2 antibody (1:100; guinea pig) (25 (link)), anti-Piwi (1:10; mouse, P4D2), anti-FLAG M2-peroxidase [horseradish peroxidase (HRP)] (1:5000; Sigma-Aldrich, #A8592), anti-H3K18Ac (1:2000; Active Motif, #39756), anti-H3K27Ac (1:2000; Active Motif, #39136), anti-H4K8Ac (1:2000; Active Motif, #61104), anti-H4K12Ac (1:2000; Active Motif, #39928), anti-guinea pig immunoglobulins-HRP (1:1000; Dako), anti-rabbit immunoglobulin G (IgG)–HRP (1:3000; Bio-Rad), anti-mouse IgG-HRP (1:3000; Bio-Rad). The chemiluminescent signals were obtained by using Chemi-Lumi One (Nacalai) and detected by Chemidoc MP Imaging system (Bio-Rad). The images were processed by using Pixelmator or Fiji. The immunoblot signals were quantified by using Fiji.

Cell pellets from selected iPSC clones were lysed in RIPA buffer (Cell Signaling Technologies) and proteins electrophoretically separated on Nu-PAGE polyacrylamide gels (Invitrogen). Proteins were transferred to nitrocellulose membranes (Invitrogen), blocked in 3% milk in TBS-Tween, and probed with antibodies to detect TPM1 (1:1000, Cell Signaling Technologies) or β-Actin (1:5000, Sigma). Secondary antibodies were anti-Rabbit IgG-HRP or anti-Mouse IgG-HRP (Bio-Rad). Western blots were imaged using a Chemi-Doc Imaging System (Bio-Rad).