Mouse eyes from 6, 12, and 24 m.o. WT and KO animals were fixed and dissected, as reported previously [18 (link)]. Briefly, under a dissection microscope, the anterior segment tissues and vitreous were removed to produce an eyecup. The retina was then gently separated from the RCSC, and four relaxing radial incisions were made in the RCSC and retina in order to produce a flat-mount. The RCSC was blocked and permeabilized overnight with a solution composed of 2.5% BSA (A9467–100G, Sigma-Aldrich) in PBS with 0.01% Triton X; the RCSC samples were then incubated with primary antibodies to N-cadherin, αSMA, and vimentin (Table 1 ). Following washing and incubation for 24 h with goat anti-rabbit IgG (H + L), Cyanine5 (A10523, 1:1000; Thermo Fisher), goat anti-rabbit IgG (H + L), and Alexa Fluor 488 (A-11034, 1:1000, Thermo Fisher), the nuclei were counterstained with DAPI 1:5000 (Sigma-Aldrich). Samples were mounted on slides with the ProLong Diamond antifade reagent. Samples that were incubated with a blocking buffer (the primary antibody was omitted) followed by secondary antibody incubation were used to correct confocal settings for a non-specific background.
Goat anti rabbit igg h l
Manufactured by Thermo Fisher Scientific
Sourced in United States
Goat anti-Rabbit IgG (H+L) is a secondary antibody used in immunoassays and other immunological techniques. It is produced by immunizing goats with purified rabbit immunoglobulin and recognizes the heavy and light chains of rabbit IgG.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse igg h l, by Thermo Fisher Scientific (22 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (11 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (7 mentions)
α 6ma, by Synaptic Systems (3 mentions)
Fluorescence microscope, by Nikon (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Enhanced chemiluminescence, by Bio-Rad (2 mentions)
Goat anti rat igg h l, by Thermo Fisher Scientific (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Caspase 1, by Cell Signaling Technology (2 mentions)
Goat anti mouse igg h l, by Jackson ImmunoResearch (2 mentions)
Flag tag (flag), by Merck Group (2 mentions)
F4 80, by Abcam (2 mentions)
Goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 conjugate, by Thermo Fisher Scientific (2 mentions)
90 protocols using goat anti rabbit igg h l
1
Flat-mount Retinal Pigment Epithelium Analysis
2
Protein Extraction and Western Blot Analysis
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Frozen colon (50 mg) was lysed in protein extraction buffer containing protease and phosphatase inhibitors (Beyotime). The samples were centrifuged at 13,000 g for 10 min at 4°C, and the protein concentration in the supernatant was determined using a BCA protein assay kit (Thermo Scientific). Protein (60 μg) was separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF; Millipore, Billerica, USA) membranes. All the primary antibodies were from Cell Signalling Technology (CST, USA) and used with the suitable dilution ratio of 1:1000 for ERK, p-ERK, P38, p-P38, JNK and GAPDH and 1:2000 for p-JNK. Membranes were incubated with appropriate secondary antibodies Goat anti-Mouse IgG (H+L) or Goat anti-Rabbit IgG (H+L) (Thermo Scientific; 1:5000). Quantification of the protein bands was performed with ImageJ (Version 1.53c; NIH, Bethesda, MD, USA) and normalized to GADPH.
3
Immunolabeling of Serum-Deprived WJ-MSCs
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Immunolabeling of control and serum-deprived WJ-MSCs were performed as per standard protocol. WJ-MSCs cultured on glass coverslips, under control and serum-deprived conditions, and were fixed in 4% PFA for 20 min at room temperature. Next, they were permeabilized using 0.1% Triton X-100. Primary antibody incubation was carried out at 4 °C overnight while secondary antibody incubation was performed at room temperature for 1 h. The primary and secondary antibodies used were anti-vitronectin (Santa Cruz Biotechnology, Inc.) and anti-vimentin (Cell Signaling Technology), and goat anti-mouse IgG H&L (Alexa Fluor® 488) pre-adsorbed (Abcam), goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibodies, and Alexa Fluor 568 (Thermo Scientific) respectively. Nucleus was labeled with DAPI (Sigma-Aldrich), following which mounting was carried out with VECTASHIELD antifade mounting medium. Images were acquired in the Zeiss Apotome module microscope using the Zen software.
4
Synthesis and Characterization of Thymidine Phosphate Analogues
5
Western Blot Analysis of Protein Lysates
6
Microglia Cytokine Secretion Assay
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ELISA kit (KMC0062, BMS607HS, BMS614INST, Thermo Fisher, USA) was used to detect the secretion of IL-6, TNF-α and IL-10 secreted by microglia. Griess assay were performed with Griess Reagent Kit (G7921, Thermo Fisher, USA). Rabbit monoclonal to CD86 (ab234401, Abcam, UK) and Rabbit polyclonal to Mannose Receptor (ab64693, Abcam, UK) were used as primary antibodies. Goat Anti-Rabbit IgG H&L (A32733, Thermo Fisher Scientific, USA) and Goat Anti-Rabbit IgG H&L (ab150079, Abcam, UK) were used as secondary antibody. Detailed methods were provided in Supporting data .
7
Kidney and Aorta Protein Analysis
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As previously described, proteins were extracted from the kidney22 and from the aorta.23 Electrophoresis was performed with 4% to 15% acrylamide Criterion gels (Bio‐Rad, Hercules, CA). Protein transfer onto nitrocellulose membranes (Bio‐Rad) was performed with the Trans‐Blot Turbo Transfer System (Bio‐Rad). Blots were blocked with Tris‐buffered saline–Tween‐BSA 5% and then incubated with the following primary antibodies: anti‐eNOS (endothelial nitric oxide synthase; BD Biosciences, Franklin Lakes, NJ; #610297), anti–phosphorylated eNOS (pS1177, BD Biosciences, #612393), anti‐CD45 (Abcam, Cambridge, UK; ab10558), anti‐NRF1 (Cell Signaling Technology, Danvers, MA; 46743s), anti‐UCP1 (Cell Signaling Technology; #14670), anti‐OPA1 (BD Biosciences; #612606), anti‐FIS‐1 (Santa Cruz Biotechnology, Dallas, TX; sc98900), anti‐p47 (BD Biosciences; #610355), anti‐gp91 (BD Biosciences; #611414) anti‐COX2 (BD Biosciences; #610204), and anti–β‐actin (Sigma, St. Louis, MO; A5316). Following incubation with a goat antirabbit IgG (H+L) or a goat antimouse IgG (H+L) secondary antibody, and the horseradish peroxidase conjugate (Thermo Fisher Scientific, Waltham, MA), the reaction was developed by enhanced chemiluminescence (Bio‐Rad) according to the manufacturer's instructions, and the signal was visualized by chemiluminescence.
8
Multicolor Immunofluorescence Staining of Tumor Sections
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Tumors were collected from the mice and snap-frozen in optimal cutting temperature medium. Tumor sections were cut using a cryotome, mounted on slides and stained with different primary antibodies, including CD8 (Abcam, cat. no. ab22378) and CD80 (Abcam, cat. no. ab100790), overnight at 4 °C following the manufacturer’s instructions. Following the addition of fluorescently labeled secondary antibodies (goat anti-rat IgG (H + L; Thermo Fisher Scientific, cat. no. A18866) and goat anti-rabbit IgG (H + L; Thermo Fisher Scientific, Cat. no. A32733)), the slides were analyzed with high-speed confocal microscopy (Andor Dragonfly 200, UK). All antibodies used in the experiments were diluted 200 times.
9
Immunofluorescent Localization of PHF14 in Cell Lines
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A549 and 293FT cells were grown on coverslips and transfected with the indicated plasmid. At 24 h after transfection, cells were washed once with PBS and fixed in 4% paraformaldehyde in PBS. Subsequently, cells were permeabilized with 0.2% Triton X-100 and treated for 30 min at room temperature with 10% BSA in PBS, followed by incubation with primary antibody for 1 h. Primary anti-PHF14 antibody (Proteintech, 24787-1-AP) was used. The secondary antibody was goat anti-rabbit IgG (H + L) conjugated with Alexa Fluor 594 (Thermo Fisher Scientific) in 1% BSA at room temperature for 20–90 min. The cells were rinsed 2–3 times with PBS (pH7.4) (5 min/wash) and then mounted with Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). The coverslips were washed extensively and fixed on slides, and representative images were obtained with a LSM810 confocal microscope using ZEN 2012 software version 8.1 (Carl Zeiss, Oberkochen, Germany).
10
Western Blot Analysis of Inflammasome Proteins
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