Formalin fixed, paraffin embedded tissue from lung and spleen samples of HIV-1 infected and uninfected mice were cut in 4 μm sections. Antigen retrieval was carried out in a Decloaking Chamber (BioCare Medical), in citrate buffer pH 6 (Invitrogen). Sections were incubated with anti-HIV-1 p24 (1:50; clone KC57 Beckman Coulter) or anti-human CD4 (1:50; clone 1F6 Abcam) antibody overnight at 4°C. Slides were washed five times in Tris buffered saline with 0.05% Tween-20 (TBST). Secondary anti-mouse HRP conjugated antibody (Dako) was applied for 1 hr at RT. After another five washes with TBST, staining was visualized using DAB substrate (Dako). Tissue samples were counterstained with Harris Modified Hematoxylin (Sigma). Whole tissue slide sections were scanned with an Olympus TissueFAXS whole slide scanning system and the number of p24+ or CD4+ cells were quantified using HistoQuest software (TissueGnostics) and by taking the average of 2 consecutive tissue sections from per sample. Background was subtracted for each sample by staining 2 consecutive tissue sections without the addition of the secondary antibody. The average number of unspecific stained cells from 2 slides were subtracted from the average number of CD4+ or p24+ cells.
Tissuefaxs whole slide scanning system
Manufactured by TissueGnostics
Sourced in United States
The TissueFAXS whole slide scanning system is a device designed to capture high-resolution digital images of entire microscope slides. It provides a comprehensive and efficient method for scanning and digitizing histological and cytological samples.
Automatically generated - may contain errors
Lab products found in correlation
Citrate buffer ph 6, by Thermo Fisher Scientific (2 mentions)
Clone 1f6, by Abcam (2 mentions)
Dab substrate, by Agilent Technologies (2 mentions)
Decloaking chamber, by Biocare Medical (2 mentions)
Histoquest software, by TissueGnostics (2 mentions)
Hrp conjugated anti mouse secondary antibody, by Agilent Technologies (2 mentions)
Harris modified hematoxylin, by Merck Group (2 mentions)
Tissuequest image analysis software, by TissueGnostics (2 mentions)
Ab237728, by Abcam (1 mentions)
Ab254415, by Abcam (1 mentions)
Ab16669, by Abcam (1 mentions)
Ab241332, by Abcam (1 mentions)
Sf100 20, by Thermo Fisher Scientific (1 mentions)
Mca497r, by Bio-Rad (1 mentions)
Sp8 laser scanning confocal microscope, by Leica (1 mentions)
Af1865, by R&D Systems (1 mentions)
D4w2z, by Cell Signaling Technology (1 mentions)
Ab1225, by Merck Group (1 mentions)
5 protocols using tissuefaxs whole slide scanning system
1
Quantification of HIV-1 Infection in Mice
2
Immunofluorescence Analysis of Sinonasal Immune Markers
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Paranasal sinus tissues of the participants were embedded in paraffin and prepared into 4 μm thick sections. After paraffin removal, the deparaffinized sections were heat-treated in citrate repair buffer (pH 6.0), followed by the addition of drops of 3% hydrogen peroxide to remove endogenous peroxidase. The sections were treated with goat serum blocking solution for 20 minutes and then reacted with the following primary antibodies for 1 hour for immunofluorescence staining: CD3 (1:1500, ab16669, Abcam), CXCR5 (1:5000, ab254415, Abcam), TIM-3 (1:1000, ab241332, Abcam), PD-1 (1:500, ab237728, Abcam). After washing, the sections were incubated with a secondary antibody (PV-6001, ZSGB-BIO) for 20 minutes. Subsequently, a rapid reaction buffer (1:100, FFBN45, DMK) was added and the sections were incubated at room temperature for 5 minutes for color development. The antibody complexes were removed by microwave treatment and the markers were counterstained. The sections were counterstained with 4′,6-diamidino-2-phenylindole at room temperature for 5 minutes. The sections were finally closed with the antifade mounting medium. After completion of staining, the sections were imaged using the TissueFAXS whole-slide scanning system (TissueGnostics, USA) and analyzed using the TissueQuest image analysis software (TissueGnostics, USA) to obtain accurate cell counts and densities.
3
Tissue Immunostaining and Whole-Slide Imaging
4
Histological and Immunofluorescence Analysis of Mouse Tissues
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Mouse tissues were fixed in 10% buffered formalin (Fisher Scientific, catalog SF100-20) overnight and embedded in paraffin. Sections (8 μm thick) were stained with H&E. Images were acquired on a TissueFAXS Whole Slide Scanning System (TissueGnostics) using a 20x objective. Nuclei quantification was performed with Image J.
For immunofluorescence staining, muscle sections were de-paraffinized in xylene and rehydrated using a graded ethanol series culminating with PBS. Following antigen retrieval using a programmable pressure cooker with “target retrieval solution”, pH 6.0 (Dako), tissue sections were blocked with 10% goat serum in PBS. The slides were then stained for CD8 (1:500, D4W2Z, Cell Signaling Technology), granzyme B (1:40, AF1865, R&D Systems), F4/80 (1:100, MCA497R, Bio-Rad), and OVA (1:500, AB1225, Millipore Sigma) for 16 hours at 4°C. Species-specific secondary antibodies conjugated to Cy5 or Cy7 fluorophores were used and incubated for one hour at room temperature in the dark. Sections were washed, counterstained with DAPI (100 ng/ml) and mounted using FluorSave (Calbiochem) mounting medium. Images were acquired on a Leica SP8 laser scanning confocal microscope using a 40x oil-immersion objective. Quantification of the fluorescent signals of the respective markers was performed using QuPath (69 (link)).
For immunofluorescence staining, muscle sections were de-paraffinized in xylene and rehydrated using a graded ethanol series culminating with PBS. Following antigen retrieval using a programmable pressure cooker with “target retrieval solution”, pH 6.0 (Dako), tissue sections were blocked with 10% goat serum in PBS. The slides were then stained for CD8 (1:500, D4W2Z, Cell Signaling Technology), granzyme B (1:40, AF1865, R&D Systems), F4/80 (1:100, MCA497R, Bio-Rad), and OVA (1:500, AB1225, Millipore Sigma) for 16 hours at 4°C. Species-specific secondary antibodies conjugated to Cy5 or Cy7 fluorophores were used and incubated for one hour at room temperature in the dark. Sections were washed, counterstained with DAPI (100 ng/ml) and mounted using FluorSave (Calbiochem) mounting medium. Images were acquired on a Leica SP8 laser scanning confocal microscope using a 40x oil-immersion objective. Quantification of the fluorescent signals of the respective markers was performed using QuPath (69 (link)).
5
Quantification of HIV-1 Infection in Mice
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Formalin fixed, paraffin embedded tissue from lung and spleen samples of HIV-1 infected and uninfected mice were cut in 4 μm sections. Antigen retrieval was carried out in a Decloaking Chamber (BioCare Medical), in citrate buffer pH 6 (Invitrogen). Sections were incubated with anti-HIV-1 p24 (1:50; clone KC57 Beckman Coulter) or anti-human CD4 (1:50; clone 1F6 Abcam) antibody overnight at 4°C. Slides were washed five times in Tris buffered saline with 0.05% Tween-20 (TBST). Secondary anti-mouse HRP conjugated antibody (Dako) was applied for 1 hr at RT. After another five washes with TBST, staining was visualized using DAB substrate (Dako). Tissue samples were counterstained with Harris Modified Hematoxylin (Sigma). Whole tissue slide sections were scanned with an Olympus TissueFAXS whole slide scanning system and the number of p24+ or CD4+ cells were quantified using HistoQuest software (TissueGnostics) and by taking the average of 2 consecutive tissue sections from per sample. Background was subtracted for each sample by staining 2 consecutive tissue sections without the addition of the secondary antibody. The average number of unspecific stained cells from 2 slides were subtracted from the average number of CD4+ or p24+ cells.
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