Mice in each group were sacrificed on the 7th day after transplantation. The liver, lung, and spleen tissues were fixed in 10% buffered formalin and embedded in paraffin blocks for subsequent hematoxylin and eosin staining. Histologic changes were graded and scored by a pathologist blinded to the clinical status of the mice. Four items were assessed as described previously [25 (link)]. To determine the infiltration of T cells in the tissues of post-HSCT mice, immunohistochemical analysis was performed on the liver, lung, and spleen tissues of recipient mice at 7 days posttransplantation, stained with the primary antibodies anti-CD4 (1 : 20, Servicebio, Wuhan, China) and anti-Cxcl7 (1 : 20, Abcam, Cambridge, UK) as well as the appropriate horseradish peroxidase-conjugated secondary antibody (1 : 50, Servicebio, Wuhan, China).
Anti cd4
Manufactured by Wuhan Servicebio Technology
Sourced in China
Anti-CD4 is a laboratory reagent used for the detection and analysis of CD4 positive cells. It is a monoclonal antibody that specifically binds to the CD4 surface antigen, which is expressed on a subset of T lymphocytes. This reagent can be utilized in various immunological techniques, such as flow cytometry, to quantify and characterize CD4+ T cells within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Anti cxcl7, by Abcam (2 mentions)
Anti cd8, by Wuhan Servicebio Technology (2 mentions)
Anti iba1, by Wuhan Servicebio Technology (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Wuhan Servicebio Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Wuhan Servicebio Technology (1 mentions)
Anti f4 80, by Wuhan Servicebio Technology (1 mentions)
Pannoramic 250 flash, by 3DHISTECH (1 mentions)
Anti ifn γ, by Bioss Antibodies (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Anti ki67, by Wuhan Servicebio Technology (1 mentions)
Anti granzymeb, by Bioss Antibodies (1 mentions)
Anti gfap, by Wuhan Servicebio Technology (1 mentions)
Anti mmp9, by Proteintech (1 mentions)
Anti il 1β, by Abcam (1 mentions)
Digital still camera, by Nikon (1 mentions)
Inverted fluorescent microscope, by Nikon (1 mentions)
Anti cd19, by Wuhan Servicebio Technology (1 mentions)
Anti il 17a, by Proteintech (1 mentions)
Anti ahr, by Abcam (1 mentions)
Anti f4 80 antibody, by Wuhan Servicebio Technology (1 mentions)
8 protocols using anti cd4
1
Histological Analysis of Post-HSCT Tissues
2
Spleen Tissue Immunostaining Protocol
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The spleen tissues were fixed with acetone. IF staining was performed using the primary antibodies anti-CD4 (1 : 200, Servicebio, Wuhan, China) and anti-Cxcl7 (1 : 2000, Abcam, Cambridge, UK) and secondary antibody conjugated to FITC (1 : 500, Servicebio, Wuhan, China) in weak light conditions. Nuclei were stained with DAPI (Servicebio, Wuhan, China). With confocal microscopy, immunostained slides were imaged.
3
Immunofluorescence Staining of Paraffin Sections
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The sections of samples were deparaffinized in xylene and rehydrated in decreasing concentrations of alcohol. After washing the sections three times in phosphate buffered saline (PBS) for 5 minutes, we blotted them with 5% goat serum for 1 hour at 37°C. The sections were incubated with the primary antibody overnight at 4°C. These sections were washed three times in PBS and then incubated with secondary antibodies for 2 hours at 37°C. The sections were subsequently rinsed three more times and stained with DAPI (Beyotime Biotechnology, Shanghai, China) for 5 minutes. Pannoramic 250FLASH (3DHISTECH, Budapest, Hungary) was used to image the results. The IF antibody and its concentration were determined for the following: anti-CXCL1 (Proteintech, 12335-1-AP, 1:100); anti-F4/80 (Servicebio, GB11027, 1:500); anti-CD86 (CST, #91882S, 1:200); anti-ARG1 (Servicebio, GB11285, 1:200); anti-CD4 (Servicebio, GB13064-2, 1:200); and anti-IFN-γ (Bioss, bs-0480R, 1:100).
4
Comprehensive Protein Expression Analysis
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Western blotting and IHC were conducted in triplicate as per previously published protocols [29 (link)]. Anti-CHSY3 for Western blot was obtained from Abcepta (Beijing, China). Anti-CHSY3, anti-Granzyme B, anti-Perforin utilized for IHC was from Bioss (Beijing, China). Anti-PD-L1, anti-CD8, anti-CD4 and anti-Ki67 were purchased from Servicebio (Wuhan, China). Anti-Alpha Tubulin obtained from Protentech (Wuhan, China).
5
Histopathological Analysis of EAE Mice
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EAE mice were sacrificed through cardiac perfusion at day 18 postinoculation. Lumbar enlargements of the spinal cord and brain were dissected and fixed in 4% paraformaldehyde for paraffin sectioning. Histopathology assays were performed as previously described [29 (link)]. Hematoxylin-eosin (H&E) staining was performed with eosin (F527FA0002, Sangon Biotech) and hematoxylin (F108FA0006, Sangon Biotech). The following antibodies were used: anti-IL-1β (Abcam, ab9722), anti-IBA1 (ServiceBio, GB12105), anti-GFAP (ServiceBio, GB11096), anti-CD4 (ServiceBio, GB13064-1), anti-MMP9 (Proteintech, 10375-2-AP), and anti-CSF1 (Affinity, DF12536). Immunostaining analyses were quantified with Image J.
6
Dual Immunofluorescence Localization of Immune Markers
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Dual immunofluorescence was performed to colocalize CD4, CD19, IL-17A and IL-22 to AhR. Briefly, optimum cutting temperature embedded skin tissues were cut into 5-μm sections and boiled in ethylenediaminetetraacetic acid buffer (pH, 9.0) for antigen retrieval. The sections were incubated with the primary antibody overnight at 4°C. The following primary antibodies (mouse antihuman) were used: anti-CD4 (Servicebio, Wuhan, China, 1:100), anti-CD19 (Servicebio, Wuhan, China, 1:100), anti-IL-17A (Proteintech, Beijing, China, 1:200), anti-IL-22 (Bioss, Beijing, China, 1:100) and anti-AhR (Abcam, Cambridge, US). Then, the sections were incubated with corresponding fluorescence secondary antibody and counterstained using 4’,6-diamidino-2- phenylindole. The sections were observed under an inverted fluorescent microscope (Nikon, Tokyo, Japan) at original magnification ×200 under appropriate absorption/emission wavelengths (Alexa Fluor, 594 nm or 488 nm). The images were captured using a Nikon Digital Still Camera and analyzed by 2 independent investigators. Immunostained cells in the dermis were counted, and results were expressed as the number of positive cells/high power field (HPF). Interobserver variations for the counts were <10%.
7
Immunohistochemical Analysis of Mouse Eye
8
Quantifying Spinal Cord Demyelination and Inflammation
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The spinal cord was dissected and xed with ice-cold 4% paraformaldehyde overnight at 4 ℃, embedded in para n, cut into 5 μm slices, and stained with luxol fast blue (LFB) and hematoxylin and eosin (HE). LFB-stained sections were scored for demyelination as follows: 0, none; 1, rare foci; 2, a few areas of demyelination; 3, one to two large areas of demyelination; and 4, extensive demyelination. HE-stained sections were also scored for in ammation as follows: 0, none; 1, a few scattered in ammatory cells; 2, perivascular in ltrates; 3, extensive perivascular cu ng with extension into adjacent parenchyma; and 4, extensive cell in ltration in the white matter [19] . Additionally, the sections were subjected to indirect immunostaining. The primary antibodies were used as follows: anti-CD4 (1:70, Servicebio, Wuhan, China), anti-MBP (1:50, Boster, Wuhan, China), and anti-Iba-1 (1:200, Servicebio). Staining was quanti ed using the HALO™ image analysis software (Indica Labs, NM, USA).
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