O.C.T. medium (Tissue Tek, Inc., Torrence, CA) embedded FP tissue was snap frozen in liquid nitrogen and stored at −70°C. Serial 4 µm sections (Frigocut 2800E, Leica Microsystem, Inc., Bannockburn, IL) were fixed in cold acetone and, after blocking with avidin and biotin (Vector Laboratories, Burlingame, CA), stained with rat anti-mouse CD4 (BD Biosciences, San Jose, CA). Biotin-labeled mouse anti-rat F(ab)2 fragments (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was applied, and immunohistochemical visualization was achieved with Vectastain Elite ABC kit (Vector), the AEC Substrate kit (Vector), and hematoxylin counterstain.
Rat anti mouse cd4
Manufactured by BD
Sourced in United Kingdom
The Rat anti-mouse CD4 is a laboratory reagent used in flow cytometry and other immunological applications. It is a monoclonal antibody that specifically binds to the CD4 antigen expressed on a subset of T lymphocytes in mice. This product can be used to identify and quantify CD4-positive cells in mouse samples.
Automatically generated - may contain errors
Lab products found in correlation
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Rat anti mouse cd8, by BD (2 mentions)
Nfatc1, by Proteintech (2 mentions)
Oct compound, by Avantor (2 mentions)
Rabbit anti mouse il 9, by Aviva Systems Biology (2 mentions)
Rabbit anti mouse pu 1, by Proteintech (2 mentions)
Nfatc2, by Proteintech (2 mentions)
Occludin, by Thermo Fisher Scientific (2 mentions)
Claudin 1, by Thermo Fisher Scientific (2 mentions)
Avidin and biotin, by Vector Laboratories (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Aec substrate kit, by Vector Laboratories (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 anti rat igg, by Abcam (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Anti mouse cd4 mab, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
16 protocols using rat anti mouse cd4
1
Immunohistochemical Staining of CD4+ Cells
2
Immunofluorescent Staining of Brain Sections
3
Immunohistochemistry of IL-17A and CD4 in Spinal Cord
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Some spinal cord tissues from each group were fixed in 4% paraformaldehyde overnight, immersed in 30% sucrose, and embedded in OCT on dry ice. The cryostat transversal lumbar spinal cord sections (20 µm) were washed twice with PBS and blocked with 10% normal mouse serum in PBS for 1 h at room temperature. The sections were incubated with goat anti-mouse IL-17A (Santa Cruz Biotechnology) or rat anti-mouse CD4 (BD PharMingen) overnight at 4°C. After being washed, the sections were incubated with PE-anti-goat IgG (Santa Cruz Biotechnology) and Alexa Fluor 488-anti-rat IgG (Abcam). Images were taken under an Olympus Confocal FV1000 microscope.
4
Cytokine Expression in T Cells
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Crude wheat gluten and ovalbumin (OVA) were obtained from Sigma (Sigma, St. Louis, MO), while gliadin was from Fluka (Sigma). Phorbol myristate acetate (PMA) and ionomycin were purchased from Sigma. The following monoclonal antibodies (mAbs) as well as isotype controls were purchased from BD Biosciences (BD Biosciences, Mountain View, CA): Alexa Fluor 488-conjugated rat anti-mouse IL-2 (JES6-5H4, IgG2b), IL-4 (11B11, IgG1), IFN-γ (XMG1.2, IgG1), FITC-conjugated rat anti-mouse IL-10 (JES5-16E3, IgG2b) and CD8 (53-6.7; IgG2a,κ), PerCP-Cy5.5-conjugated hamster anti-mouse CD3 (145-2C11; IgG1,κ), rat anti-mouse CD4 (RM4-5; IgG2a,κ), CD8 (53-6.7; IgG2a,κ) and PE-conjugated hamster anti-mouse γδ T cell receptor (GL3; IgG2,κ) mAbs. Mouse Treg staining kit Cat.No. 88–8111, PE-conjugated rat anti-mouse Foxp3 mAb (FJK-16s; IgG2a,κ) and FITC-conjugated rat anti-mouse CD4 mAb (RM4-5; IgG2a,κ) were from eBioscience (eBioscience, San Diego, CA). The anti-mouse CD4 mAb (BD Biosciences) was used in combination with intracellular cytokine staining using Cytofix/Cytoperm kit (BD Biosciences), while the anti-mouse CD4 mAb (from the eBioscience kit no. 88–8111) was used when detecting Foxp3+CD4+ T cells (with no prior PMA inomycin stimulation) by using the Treg staining kit 88–8111.
5
Histology and Immunofluorescence Staining
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For histology, sections were stained with hematoxylin and eosin (H&E), according to established protocols. For immunofluorescence, the sections were air-dried at room temperature for 15 min and with 4% paraformaldehyde for 10 min. Sections were then rinsed with 1 X PBST (0.25% Triton-X100). Blocking was performed for 30 min at room temperature in 5% normal serum in PBS. Sections were incubated with primary antibodies diluted in PBS for 2 h at room temperature. Sections were washed with 1 X PBS and incubated with secondary antibodies diluted in PBS for 1 h at room temperature. After washing again, sections were mounted with the VECTASHIELD HardSet mounting medium with DAPI (Vector Laboratories, Burlingame, CA) and imaged. Primary antibodies used were: rat anti-mouse Ly6G and rat anti-mouse F4/80 (BioLegend, San Diego, CA), rat anti-mouse CD31, rat anti-mouse CD4, and rat anti-mouse CD8a (BD PharMingen, Mississauga, ON). Alexa Fluor 546, goat anti-rat (Life Technology, Burlington, ON) was used as the secondary antibody. All histology and immunofluorescence images are representative of at least three independent animal experiments.
6
Multiparameter Flow Cytometry of Immune Cells
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BAL fluid, lungs, and mediastinal LNs were analyzed by FACS (FACSCalibur BD Biosciences). The following mAbs were used for T cell analysis: rat anti-mouse CD4, rat anti-mouse CD8a, anti-CD3e, allophycocyanin-conjugated rat anti-mouse CD25, anti-CD62L, and anti-TCR Vβ5.1/5.2 (all from BD Pharmingen). Granulocytes in the BAL were detected using rat anti-mouse GR-1 (BD Pharmingen).
7
Immunofluorescent Detection of Th17 Cells
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To detect Th17 cells in the tissues, rabbit anti-IL-17 (Abcam, Cambridge, UK) and rat anti-mouse CD4 (BD Biosciences, San Diego, CA, USA) were used as primary antibodies. Either rabbit IgG (Abcam) or rat IgG2a (BD Biosciences) was used as isotype control. Briefly, 8 μm sections were fixed with cold acetone for 5 min and blocked with 5% goat serum in PBS with 0.1% tritonX-100. The sections were then incubated with primary antibodies at 4 °C. After incubation overnight, the sections were washed with PBS three times, and then incubated with secondary antibodies, FITC-conjugated rabbit IgG (Sigma), and Alexa-594-conjugated Rat IgG (Invitrogen). Nucleus staining with Hoechst 33258 (Invitrogen) was performed as counterstaining. Tissues were visualized using a fluorescence microscope.
8
Quantifying Immune Cells in Kidney
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Immunoperoxidase staining to detect CD68 positive macrophages and CD4+ T-cells was performed on frozen PLP-fixed sections using the Polink-2 HRP Plus kit (Newmarket Scientific). Primary antibodies used were rat anti-mouse CD68 (Serotec) and rat anti-mouse CD4 (Pharmingen). For intra-glomerular scoring, only macrophages or CD4+ cells inside glomeruli, as defined by Bowman’s capsule, were counted. 25 glomeruli were scored per kidney. For interstitial macrophage and CD4+ cell scoring, whole sections were inspected and all kidneys from a single experiment were ranked in order of increasing interstitial macrophage or CD4+ staining. Sequential ascending scores were then assigned for all sections.
9
Multicolor Flow Cytometry of Splenocytes
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The expression of CD4+, CD8+, CD11b+, Gr-1+, and B220+ by splenocytes was measured by flow cytometry. Briefly, splenocytes were stained with rat anti-mouse CD4 (BD Biosciences, San Jose, CA) and Gr-1 (eBioscience, Waltham, MA) conjugated with FITC and/or rat anti-mouse CD8 (BD Biosciences, San Jose, CA) and B220 (BD Biosciences, San Jose, CA) conjugated with PE-Cy5 and/or rat anti-mouse CD11b (eBioscience, Waltham, MA) conjugated with APC antibodies in staining buffer (PBS containing 2% FBS and 0.09% sodium azide) avoiding light on ice for 30 min. Appropriate rat anti-mouse antibodies were applied as the isotype control for evaluating non-specific binding. After washing, the single-cell fluorescence of 10,000 cells for each sample was measured by a flow cytometer (BD FACSCalibur, San Jose, CA) Data was analyzed by Flowjo 10.4 software (FlowJo LLC, Ashland, OR).
10
Immunohistochemical Analysis of Mouse Cornea
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The eyes and lids or CLNs of mice in each group were excised, embedded in optimal cutting temperature (OCT) compound (VWR, Suwanee, GA), and flash-frozen in liquid nitrogen. Cryosections from mouse globes or CLNs were cut with a cryostat (HM 500; Micron, Waldorf, Germany), and stored at −80 °C before use. Cultured CD4+ T cells were fixed in acetone for staining. For whole mount IF staining, the treated corneal and conjunctival tissues were fixed in 10% formaldehyde for 5 minutes at room temperature (RT) and in acetone for 3 minutes at −20 °C. IHC or IF staining was performed as previously described respectively.11 (link), 50 (link)The primary antibodies used for this study included: rat anti-mouse CD4 from BD Pharmingen (San Jose, CA); rabbit anti-mouse IL-9 from Aviva Systems Biology (San Diego, CA); rabbit anti-mouse IL-9R, IL-33, ZO-1, claudin 1 and occludin from Thermo Fisher Scientific (Waltham, MA); rabbit anti-mouse PU.1, NFATc1, or NFATc2 from Proteintech Group (Rosemont, IL); and rabbit anti-mouse ST2, E-cadherin from Novus Biologicals (Centennial CO). The quantitative analysis for barrier structure was performed by measuring the IF length of each apical junction protein on the images using ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/ , 1997–2018).
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