Cells were fixed using 4% paraformaldehyde (Alfa Aesar, 43368) for 10 min and incubated in blocking PBS solution containing 2% BSA (AppliChem, 9048-46-8) and 0.2% Triton X-100 (Sigma-Aldrich, 9002-93-1) for 30 min to permeabilize cell membrane and block unspecific antigen binding. As the next step, the cells were incubated with primary antibodies in blocking solution for 2 hours, followed by 30 min of incubation with secondary antibodies and 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining (1:1000; Thermo Fisher Scientific, 62248). Stained cells were imaged using a Nikon microscope (ECLIPSE Ti2). The following primary antibodies have been used in this study: anti-human/mouse/rat/chicken Pax7 (5 μg/ml; R&D Systems, MAB1675), anti-human/mouse Myod1 (5.8A) (1:100; Thermo Fisher Scientific, MA512902), anti-mouse MyHC (1:1000; R&D Systems, MAB4470), and anti–Ki-67 (SP6) (1:250; Thermo Fisher Scientific, MA514520). The following secondary antibodies were used in this study at 1:500 dilution: anti-mouse immunoglobulin G1 (IgG1; goat, Alexa Fluor 647) (Thermo Fisher Scientific, A21240), anti-mouse IgG2B (goat, Alexa Fluor 546) (Thermo Fisher Scientific, A21240), and anti-rabbit IgG (H + L) (donkey, Alexa Fluor 546) (Thermo Fisher Scientific, A10040).
Anti rabbit igg h l
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-rabbit IgG (H+L) is a secondary antibody that binds to the heavy and light chains of rabbit immunoglobulin G (IgG). It is commonly used in immunoassays and other applications that require the detection or isolation of rabbit antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (7 mentions)
Anti mouse igg h l, by Thermo Fisher Scientific (6 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Anti ki67 sp6, by Thermo Fisher Scientific (1 mentions)
Anti mouse igg2b, by Thermo Fisher Scientific (1 mentions)
Eclipse ti2, by Nikon (1 mentions)
Goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
P mek1 2 ser217 221, by Cell Signaling Technology (1 mentions)
Anti biotin hrp linked antibody, by Cell Signaling Technology (1 mentions)
P akt ser473 d9e xp, by Cell Signaling Technology (1 mentions)
Immobilon western chemiluminescent horseradish peroxidase hrp substrate kit, by Merck Group (1 mentions)
Cd133, by Miltenyi Biotec (1 mentions)
Mek1 2, by Cell Signaling Technology (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Nacalai Tesque (1 mentions)
Rabbit anti laminin, by Merck Group (1 mentions)
Anti ctnt, by Abcam (1 mentions)
Rabbit anti vimentin, by Abcam (1 mentions)
Rabbit anti collagen type 1, by Abcam (1 mentions)
17 protocols using anti rabbit igg h l
1
Immunofluorescence Staining of Muscle Cells
2
Quantification of Protein Expression by Western Blot
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Western blot analysis was performed as previously described38 (link). Briefly, 30 or 40 μg of total protein were separated by 10% denaturing SDS–PAGE and transferred onto 0.45 μm nitrocellulose membranes overnight. The membranes were incubated with primary ß-Catenin (6B3) (1:1000, Cell Signaling, #9582), CD133 (1:250, Miltenyi, #130-092-395), p-AKT (Ser 473) (D9E) XP (1:1000, Cell Signaling, #4060), AKT (1:1000, Cell Signaling, #9272), p-MEK1/2 (Ser217/221) (1:1000, Cell Signaling, #9121), or MEK1/2 (1:1000, Cell Signaling, #9122) antibodies overnight at 4 °C. Secondary horseradish-peroxidase-coupled antibodies goat-anti-mouse IgG (H + L) (1:10,000,Thermo Fisher Scientific) or anti-rabbit IgG (H + L) (1:10,000, Thermo Fisher Scientific), and anti-biotin, HRP-linked antibody (1:5000, Cell Signaling) were then applied for 1 h at RT. Hybridization with GAPDH-HRP (6C5) (1:10,000–20,000, Abnova, #MAB5476) coupled antibody was performed for 30 min at RT as a housekeeping gene. Antibodies were visualized using the Immobilon Western Chemiluminescent horseradish peroxidase (HRP) substrate kit (Merck Millipore). Images were generated using Gene Genome Syngene Bio Imaging and quantified using Image J (Rasband, W.S., U.S. National Institutes of Health).
3
Western Blot Analysis of PARP, Caspase-3, and β-Tubulin
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Total protein was extracted using RIPA lysis buffer (Beyotime, cat no. P0013C) containing 1% PMSF. Protein samples were separated via SDS PAGE and then transferred to polyvinyl difluoride membranes. After blocking with 5% bovine serum albumin, the membranes were incubated overnight at 4°C with primary antibody against PARP (1:1000 dilution, Epitomics, cat no. 1078-S), casepase3 (1:1000 dilution, Epitomics, cat no. 1087-1) and β-Tubulin (1:1000 dilution, Abcam, ab6046), and followed by incubation with peroxidase conjugated secondary antibodies (anti-rabbit IgG, (H+L) (Thermo, cat no. 31460) or anti-mouse IgG, (H+L) (Thermo, cat no. 31430)). The immunoreactive bands were then visualised with enhanced chemiluminescence (ECL) (Thermo, cat no. 32209).
4
Cardiac Tissue Immunofluorescence Imaging
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Cardiac tissues were fixed with 4% paraformaldehyde (Nacalai Tesque). Experiments were performed on paraffin sections after antigen retrieval with citrate buffer (pH 6.0) at 121°C for 20 min. The tissues were incubated with primary antibodies including mouse or rabbit anti-cTnT (1:200 dilution; Abcam, Cambridge, UK), rabbit anti-vimentin (1:200, Abcam), mouse anti-sarcomeric α-actinin (1:400; Sigma-Aldrich), rabbit anti-connexin43 (1:100; Abcam), mouse anti-MLC2a (1:100; Synaptic Systems, Goettingen, Germany), rabbit anti-MLC2v (1:200; Proteintech, Rosemont, IL, USA), mouse anti-FN (1:200; Abcam), rabbit anti-laminin (1:30; Sigma-Aldrich), and rabbit anti-collagen type I (1:500; Abcam). The endothelial cells were immunostained with mouse anti-CD31 antibody (1:200; Dako, Glostrup, Denmark). The tissues were permeabilized with 0.2% Triton X (Sigma-Aldrich), and then non-specific reactivity was blocked with 1% BSA. The tissues were labeled by primary antibodies. Secondary antibodies (1:200) such as Alexa Fluor 488- or Alexa Fluor 546-conjugated goat anti-mouse or anti-rabbit IgG (H+L) (Thermo Fisher Scientific) were added to the tissues. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies) and observed by confocal laser scanning microscopy (FLUOVIEW FV10i; Olympus, Tokyo, Japan).
5
Immunostaining of Harpegnathos Brains
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Harpegnathos brains were dissected in DMEM and fixed in 4% paraformaldehyde overnight at 4°C. Fly heads were dissected in 1× PBS and fixed in 4% paraformaldehyde for 2 hours at room temperature. Washed in PBS twice and embedded with 4% agarose gel in PBS. Thick sections (100 μm) were obtained with a Vibratome (Leica VT1000S), permeabilized in PBST (1× PBS + 0.5% Triton X-100) two times for 10 min, and then blocked with 5% goat serum in PBST for at least 1 hour at room temperature. Sections were incubated with primary antibodies overnight at 4°C in 5% goat serum in PBST, washed in PBST three times for 20 min, incubated with appropriate fluorescently labeled secondary antibodies for 2 hours in PBST, washed six times with PBST, and stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min. Last, samples were washed three times for 5 min and then mounted in antifade mounting medium (Vector Laboratories, H-1000). Sections were imaged with a Leica SPE laser scanning confocal microscope. Antibodies used were as follows: anti-Synapsin: DSHB 3C11 (1:20); anti–Pka-C1: Cusabio (1:1000); anti-egr (1:500, a gift from M. Miura) (59 (link)); anti-mouse immunoglobulin G (IgG) (H+L), Alexa Fluor 488: ThermoFisher Scientific (1:500), anti-rabbit IgG (H+L), Alex Fluor 568: ThermoFisher Scientific (1:500).
6
Transient Expression of CyRPA and P113
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Plasmids containing the CyRPA and P113 coding sequences were chemically synthesized as described above for the merozoite cell surface library but with three differences: constructs included their endogenous signal peptides, the GPI-sequence of P113 was retained, and the proteins lacked any protein tags; these plasmids were transiently transfected in HEK293 cells. After 24 and 48 h the culture supernatant and cells were collected, probed with the appropriate polyclonal IgG raised against CyRPA or P113 and stained with anti-rabbit IgG (H+L) conjugated to Alexa Fluor 568 (ThermoFisher Scientific Cat. No. A10042). PBS was used to wash the cells between antibody incubations. Flow cytometry was performed on a BD LSR Fortessa.
7
Quantitative Analysis of Lentiviral Proteins
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Concentrated lentivirus or LVLPs (400 ng p24 by ELISA) were lysed in 60 μl of 1× Laemmli sample buffer. The proteins in each sample were separated on SDS-PAGE gels and analyzed by western blotting. The antibodies used include mouse monoclonal anti-SaCas9 antibody (Millipore Sigma, MAB131872, clone 6F7, 1:1000), rabbit polyclonal HIV1 p17 antibody for matrix protein (MA, ThermoFisher Scientific, Cat No. PA1–4954, 1:1000), rabbit polyclonal HIV1 p15 antibody for nucleocapsid protein (NC, Abcam, Cat No. ab66951, 1:1000) and p24 mouse monoclonal antibody for capsid protein (CA, Cell Biolabs, Cat No. 310810, 1:1000). HRP conjugated anti-Mouse IgG (H+L) (ThermoFisher Scientific, Cat No. 31430, 1:5000) and anti-Rabbit IgG (H+L) (Cat No. 31460, 1:5000) secondary antibodies were used in western blotting. Cas9 RNP standards were from BioVision Incorporation (Cat# M1280–50). Chemiluminescent reagents (Pierce) were used to visualize the protein signals under the LAS-3000 system (Fujifilm). Densitometry (NIH ImageJ) was used to quantitate protein amount.
8
Quantifying Bcl-2 Expression in H209 Cells
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Paraffin‐embedded H209 cells were treated as described above. The cells were stained with anti‐TTF‐1 and anti‐Bcl‐2 antibodies. Stained cells were visualized using anti‐mouse IgG H&L (Alexa Fluor 594; Thermo Fisher Scientific), anti‐rabbit IgG H&L (Alexa Fluor 488; Thermo Fisher Scientific), and DAPI. Images were captured with the all‐in‐one fluorescence microscope BZ‐X710. The expression of Bcl‐2 was quantified by area fraction measurement of ImageJ and normalized by cell number. For each condition, randomly selected two enlarged images were used for calculation.
9
Coimmunoprecipitation of PIAS2 and NP
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The coimmunoprecipitation assay was performed in HEK 293T cells systems as a previous study (Cheng et al., 2015b (link)). In brief, HEK 293T cells were cultured until 80% confluence and then transfected with duPIAS2-HA and DK212-NP-Flag using Hieff Trans. At 36 h post-transfection, the cells were washed with cold PBS and lysed with an IP Western lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA (Beyotime, China), and 1% PMSF (Yeasen, China) for 20 min on ice. The cell lysate was centrifuged at 14,000 g at 4 °C for 15 min. The supernatant was incubated with 50 μL agarose beads (Thermo Fisher Scientific, United States) and rocked for 6 h at 4°C. The beads were washed with TBST three times after incubation and boiled in 2∗SDS Buffer (Dingguo, China) for 10 min. Protein samples were subjected to Western blot. NP-Flag and duPIAS2-HA were detected using anti-NP mouse monoclonal antibodies (Sino Biological, China) and anti-HA rabbit polyclonal antibodies (Sigma, United States), respectively. The secondary antibodies were DyLight 800 goat antimouse and antirabbit IgG (H+L) (Thermo Fisher Scientific, United States). The membranes were imaged using an Odyssey infrared imaging system (Li-CoR, United States).
10
Immunofluorescence Analysis of Smooth Muscle Cell Markers
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