Detailed methods are described as previously.22 Briefly, 5 μm sections were prepared. Slices were deparaffinized and incubated with 5% bovine serum albumin for 30 minutes. Afterwards, samples were incubated with primary antibody at 4°C overnight. After washing with PBS, the sections were incubated for with secondary antibody for 2 hours. Fields of connective tissue adjacent to the junctional epithelium as well as alveolar bone were selected from each sample to calculate the number of positive cells. Primary antibodies used were as follows: Rat Anti‐CD68 antibody (1:100, Abcam, ab53444) and Rabbit Anti‐CD206 antibody (1:200, Abcam, ab64693). Secondary antibodies used were as follows: Goat Anti‐Rat IgG H&L (1:1000, Abcam, ab150157) and Goat Anti‐Rabbit IgG H&L (1:2000, Abcam, ab150079).
Goat anti rat igg h l
Manufactured by Abcam
Sourced in United States
Goat Anti-Rat IgG H&L is a secondary antibody that binds to the heavy and light chains of rat immunoglobulin G (IgG). It is produced in goats and can be used in various immunoassay techniques such as ELISA, Western blotting, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg h l, by Abcam (7 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Goat anti chicken igy h l, by Abcam (2 mentions)
Donkey anti mouse igg h l, by Thermo Fisher Scientific (2 mentions)
Donkey anti rabbit igg h l, by Abcam (2 mentions)
Reddot1 far red nuclear stain, by Biotium (2 mentions)
Rabbit anti lyz, by Agilent Technologies (2 mentions)
Rabbit anti olfm4, by Cell Signaling Technology (2 mentions)
Recombinant rabbit anti aldolase b aldolase c, by Abcam (2 mentions)
Mouse anti human krt20, by Agilent Technologies (2 mentions)
Mouse anti chr a, by Santa Cruz Biotechnology (2 mentions)
Rat anti e cadherin, by Santa Cruz Biotechnology (2 mentions)
Cd24 monoclonal antibody, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit igg h l alexa fluor 405, by Abcam (2 mentions)
Wga conjugated to cf488a, by Biotium (2 mentions)
Sytox orange nucleic acid stain, by Thermo Fisher Scientific (2 mentions)
Mouse anti ki67, by BD (2 mentions)
Goat anti mouse igg h l, by Abcam (2 mentions)
Ab53444, by Abcam (1 mentions)
16 protocols using goat anti rat igg h l
1
Immunohistochemical Analysis of Inflammatory Cells
2
Immunohistochemical Analysis of Tumor Microenvironment
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Tumors from mice were immobilized in 4% paraformaldehyde for 48 h and then metastasized into 75% alcohol. The thin sections of 3 μm were obtained by microtome and stained with different primary antibodies: CD8 (Thermo Fisher Scienctific, cat. no. MA1-81180), CD80 (Abcam, cat. no. ab254579), and F4/80 (Cell Signaling technology, cat. no. 71299S), CD47 (ABclonal, cat. no. A7278) overnight at 4 °C following the manufacturer instructions. Following the addition of fluorescently labelled secondary antibodies (goat anti-rat IgG (H&L; Abcam, cat. no. ab150159) and goat anti-rabbit IgG (H&L; Abcam, cat. no. ab150077)), the slides were analyzed with a confocal microscope. The antibodies used in the experiment were diluted according to instructions.
3
Immunofluorescent Analysis of Endothelial and Cartilage Markers
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The sections were fixed with 4% paraformaldehyde for 30 min, permeabilized in PBS containing 1% Triton X-100 for 10 min, followed by washing three times with PBS, and blocked with 10% goat serum in PBS for 30 min. Then, the sections were incubated with primary antibodies against CD31 (abcam, UK) or collagen type II (abmart, China) in the Superblock solution overnight at 4 °C. On the next day, sections were washed with PBS three times for 5 min each, followed by incubation with the Goat Anti-Rabbit IgG (abcam, UK) or Goat Anti-Rat IgG H&L (abcam, UK) for 2 h at room temperature. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) for 2 min. After washing, the tissue was observed under the CLSM in the darkroom. Quantification for CD31 and COL II intensities were determined based on the obtained mHIC images via ImageJ software.
4
Immunocytochemical Characterization of Cell Cocultures
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Cocultures were fixed with 4% paraformaldehyde (Thermo Fisher Scientific) for 20 min at room temperature and then treated with 0.2% Triton X-100 for 15 min. Subsequently, coculture samples were incubated in 4% bovine serum albumin (BSA; Millipore, Burlington, MA, USA) at 4°C overnight. Primary antibodies diluted in 1% BSA were added to cells, and the mixture was incubated at 4°C overnight. Cells were then incubated with secondary antibodies at room temperature for 2 hours. The primary antibodies used were anti–c-Jun (1:500; Abcam), anti-MBP (1:500; Abcam), and anti-TuJ1 (1:1000; Abcam). The secondary antibodies used were goat anti-rabbit immunoglobulin G (IgG) heavy and light chains (H&L) (1:500 to 1:1000; Abcam), goat anti-rat IgG H&L (1:500; Abcam), and goat anti-chicken IgY H&L (1:500; Abcam). Last, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies, Carlsbad, CA, USA) for 15 min. All images were acquired using an inverted confocal laser scanning microscope (LSM 700) equipped with solid-state lasers (405, 488, 555, and 649 nm).
5
Multicolor Immunostaining Protocol
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The primary antibodies used in this study are as follows: rabbit anti-Lyz (1:800; Dako, #A0099), rabbit anti-Olfm4 (1:500; Cell Signaling Technology, #39141), recombinant rabbit anti-aldolase B + aldolase C (1:300; Abcam, #ab75751), mouse anti-human KRT20 (1:500; Dako, #M701929-2), mouse anti–Chr-A (1:50; Santa Cruz Biotechnology, #sc-393941), rat anti–E-cadherin (1:400; Santa Cruz Biotechnology, #sc-59778), CD24 Monoclonal Antibody (1:200; Thermo Fisher Scientific, #17-0242-80), and mouse anti-Ki67 (1:200; BD Biosciences, 550609). The secondary antibodies used in this study are as follows: Goat Anti-Rabbit IgG H&L (Alexa Fluor 405) preadsorbed (1:1000; Abcam, #ab175654), Goat Anti-Rat IgG H&L (Alexa Fluor 555) preadsorbed (1:1000; Abcam, #ab150166), Donkey Anti-Mouse IgG H&L (Alexa Fluor 647) (1:500; Thermo Fisher Scientific, #A31571), and Donkey Anti-Rabbit IgG H&L (Alexa Fluor 405) preadsorbed (1:1000; Abcam, #ab175649). The dyes used in this study are as follows: WGA conjugated to CF488A (5 μg/ml; Biotium), RedDot1 Far-Red Nuclear stain (1:200; Biotium), and SYTOX Orange Nucleic Acid Stain (1:5000; Thermo Fisher Scientific, #S11368). The order of staining, optimized to ensure good staining quality for all cell types, was based on the staining quality and stripping difficulty of each antibody (fig. S1G).
6
Immunofluorescence and Immunohistochemistry Protocols
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The slices from ankle joint samples were incubated with anti-F4/80 (Abcam, cat. # ab6640) and anti-iNOS (Abcam, cat. # ab15323) primary antibodies overnight at 4°C. The secondary antibody included Goat-anti-Rat IgG H&L (Alexa Fluor® 647; Abcam. cat. # ab150167) and Goat-Anti-Rabbit IgG H&L (Alexa Fluor® 488; Abcam. cat. # ab150077). And the nuclei were stained by DAPI (4',6-diamidino-2-phenylindole, sigma, cat. # D9564). The images were recorded by an Olympus BX-51 microscope.
For immunofluorescence, PEMs were seeded on round coverslip in 24-well plates. After stimulation at certain time points, cells were fixed by 4% paraformaldehyde for 10 min, permeabilized by 0.5% Triton-X, and blocked by 3% Bovine Serum Albumin (BSA) for 30 min at room temperature. The cells on the coverslip were incubated with anti-NF-κB p65 antibody (Abcam, cat. # ab16502) overnight at 4°C and goat anti-rabbit IgG Alexa Fluor® 488 (Abcam. cat. # ab150077) for 1 h at room temperature. The nuclei were stained by Bisbenzimide H 33342 Trihydrochloride (Hoechst; Sigma Aldrich, CAS: # 23491-52-3). Images were recorded by an Olympus BX-51 microscope.
Immunohistochemistry was performed in accordance with the kit's instructions (Zhongshan Jinqiao PV-9000 Universal Two-Step Test Kit) and the slices were incubated with anti-CD3 antibody (Abcam, cat. # ab56313) overnight at 4°C.
For immunofluorescence, PEMs were seeded on round coverslip in 24-well plates. After stimulation at certain time points, cells were fixed by 4% paraformaldehyde for 10 min, permeabilized by 0.5% Triton-X, and blocked by 3% Bovine Serum Albumin (BSA) for 30 min at room temperature. The cells on the coverslip were incubated with anti-NF-κB p65 antibody (Abcam, cat. # ab16502) overnight at 4°C and goat anti-rabbit IgG Alexa Fluor® 488 (Abcam. cat. # ab150077) for 1 h at room temperature. The nuclei were stained by Bisbenzimide H 33342 Trihydrochloride (Hoechst; Sigma Aldrich, CAS: # 23491-52-3). Images were recorded by an Olympus BX-51 microscope.
Immunohistochemistry was performed in accordance with the kit's instructions (Zhongshan Jinqiao PV-9000 Universal Two-Step Test Kit) and the slices were incubated with anti-CD3 antibody (Abcam, cat. # ab56313) overnight at 4°C.
7
Western Blot Analysis of Apoptosis and STAT Signaling
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The cells were mixed with RIPA lysis buffer to extract total protein. The protein concentration was measured by bicinchoninic acid (BCA) method. Then protein samples (50 µg) were subjected to 10% SDS-PAGE gel electrophoresis and transferred to PVDF membranes. Subsequently, PVDF membranes were blocked in 5% skimmed milk for 1 h at room temperature and then incubated with primary antibody overnight at 4°C. Cleaved-caspase 3 (cat. no. ab49822, 1:1,000), p-STAT3 (cat. no. ab76315, 1:3,000), t-STAT3 (cat. no. ab68153, 1:2,000), Bcl-2 (cat. no. ab182858, 1:2,000), Bcl-XL (cat. no. ab32370, 1:1,000), GAPDH (cat. no. ab8245, 1:5,000), p-STAT1 (cat. no. ab109461, 1:3,000), t-STAT1 (cat. no. ab180814, 1:1,000), p-STAT2 (cat. no. ab53132, 1:3,000), t-STAT2 (cat. no. ab32367, 1:2,000), p-STAT5 (cat. no. ab32364, 1:2,000), t-STAT5 (cat. no. ab32364, 1:1,000) were purchased from Abcam. Next day, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG H&L (cat. no. ab205718; 1:6,000, Abcam) or goat anti-rat IgG H&L (cat. no. ab97057; 1:5,000, Abcam) secondary antibodies at room temperature for 2 h. Finally, the protein bands were detected using the Tanon-5200 Multi chemiluminescent gel imaging system.
8
Histological Analysis of Mouse Intestine
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Intestine tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm sections and dewaxed, rehydrated, and then stained with hematoxylin & eosin or AB/PAS. For immunohistochemistry of Gr-1 and F4/80, cryostat sections (10 μm thick) were blocked with 3% H2O2 and then with 5% goat serum in PBST (PBS with 0.1% Triton X 100). For immunohistochemistry of Ki67 and lysozyme, paraffin-embedded sections (8 μm thick) were dewaxed and rehydrated, before incubated in 0.01 M sodium citrate buffer (pH 6.0) for heat-induced antigen retrieval. The sections were blocked with 3% H2O2 and then with 5% goat serum in PBST (PBS with 0.1% Triton X 100) and incubated with primary antibodies at 4 °C overnight and then washed 3 times with PBST before incubating with secondary antibodies. The signals were detected by SignalStain® DAB Substrate kit (CST, 8059). Primary antibodies for immunohistochemistry used: anti-Ki67 (CST, 12202), anti-F4/80 (Biolegend, 123102), anti-Gr-1 (BD, 550291) and lysozyme (Invitrogen, PA5-16668). Secondary antibodies for immunohistochemistry used: goat anti-rat IgG H&L (Abcam, ab214882), goat anti-rabbit IgG H&L (Abcam, ab214880).
9
Immunofluorescent Staining of F4/80 in Tissue
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Tissue sections were fixed in 4% PFA for 15 min and permeabilized with 0.25% Triton X-100 in PBS for another 15 min. After being blocked with 3% bovine serum albumin (BSA) in PBS for 1 h, sections were probed with F4/80 (BioLegend, 123101, 1:100) primary antibody overnight. Then, sections were washed and probed with Goat Anti-Rat IgG H&L (Alexa Fluor 568, Abcam, ab175476, 1:300) for 2 h. Nuclei were counterstained with DAPI for 5 min. Images of the stained samples were captured with Leica TCS-SP8 and Leica TCS-SP8 DIVE (Leica Microsystems, Wetzlar, Germany).
10
Immunofluorescence and Western Blot Analysis
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DMN was purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). The primary rabbit anti-mouse polyclonal antibodies against α-smooth muscle actin (α-SMA; catalog no. ab66133), C-X-C chemokine receptor type 4 (CXCR4; catalog no. ab2074), extracellular signal-regulated kinase (ERK1/2; catalog no. ab17942) and nuclear factor κB (NF-κB) p65 subunit (NF-κBp65; catalog no. ab16502), a rabbit anti-mouse monoclonal antibody against β-catenin (catalog no. ab32572), a rat anti-mouse monoclonal antibody against cluster of differentiation (CD) 90 (catalog no. ab3105), rabbit anti-rat polyclonal antibodies against albumin (Alb; catalog no. ab135575) and cytokeratin (CK) 18 (catalog no. ab189444), and the secondary antibodies goat anti-rat IgG H&L (Alexa Fluor® 488; catalog no. ab150157) and goat anti-rabbit IgG H&L (Alexa Fluor® 647; catalog no. ab150079), were purchased from Abcam (Cambridge, MA, USA). The primary antibody against β-actin (catalog no. bs-0061R) was obtained from BIOSS (Beijing, China). The secondary antibody peroxidase-conjugated AffiniPure goat anti-rabbit IgG (catalog no. ZB-2301) was obtained from ZSGB-BIO (Beijing, China). The primers, Takara Mini BEST Universal RNA Extraction kit (catalog no. 9767), PrimeScript™ RT Master mix (catalog no. RR036A) and SYBR® Premix Ex Taq™ (catalog no. RR420A) were obtained from Takara Bio, Inc. (Otsu, Japan).
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