Immunohistochemistry for CD4, CD8, and Foxp3 was performed as follows. Each tissue sample from the right lower limb containing the AT was embedded in paraffin and sectioned at 4 μm thickness. The following primary antibodies were used: anti-CD4 rabbit polyclonal antibody (Bioss, Woburn, MA, USA; bs-0647R; 1:200), anti-CD8 rabbit polyclonal antibody (Biorbyt Ltd., Cambridge, UK; orb10325; 1:100), and anti-Foxp3 rabbit polyclonal antibody (Novus Biologicals, Colorado, USA; NB100-39002; 1:200). Anti-mouse or rabbit IgG conjugated with peroxidase-labeled polymers (EnVision, Shanghai, China; Dako, Carpinteria, CA, USA) was used as the secondary antibody. The number of infiltrating immune-related cells surrounding the ATs was measured in the Control, CA, PD, and CA + PD groups (n = 8 for each group). Photographs of the ATs were captured from the four groups. For each analysis, five independent HPFs of the AT region were randomly selected per mouse. Observations were performed using a BZ-9000 microscope (KEYENCE, Osaka, Japan). The slides were coded and the positive cells for each marker were counted by two examiners who were blinded to the slides. All the mice were evaluated with the same resolution and clarity. The mean value of the positive cells in one HPF of each mouse was summed and divided by the total number of mice to obtain the mean value and standard deviation.
Nb100 39002
Manufactured by Novus Biologicals
Sourced in United States
NB100-39002 is a laboratory equipment product offered by Novus Biologicals. It is designed to perform a specific core function, but further details on its intended use are not provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab207082, by Abcam (2 mentions)
Blocking one histo, by Nacalai Tesque (2 mentions)
Dako target retrieval solution, by Agilent Technologies (2 mentions)
Sc 21763, by Santa Cruz Biotechnology (2 mentions)
Sc 268, by Santa Cruz Biotechnology (2 mentions)
Bz 9000 microscope, by Keyence (1 mentions)
Ab282110, by Abcam (1 mentions)
Peroxidase stain 3 3 diaminobenzidine dab kit, by Nacalai Tesque (1 mentions)
Ab203035, by Abcam (1 mentions)
Ab183685, by Abcam (1 mentions)
Axioskop 40 light microscope, by Zeiss (1 mentions)
Ab32197, by Abcam (1 mentions)
Ab125212, by Abcam (1 mentions)
Bs 1035r, by Bioss Antibodies (1 mentions)
Streptavidin apc, by BioLegend (1 mentions)
Immpact dab, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Bz x810, by Keyence (1 mentions)
Ph 6.0 target retrieval solution, by Agilent Technologies (1 mentions)
8 protocols using nb100 39002
1
Immunohistochemical Quantification of Immune Cell Infiltration
2
Immunohistochemical analysis of T-cell lineage transcription factors
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After deparaffinization and rehydration, tissuesections were treated with sodium citrate buffer (0.01 M) in a pressure cooker for 2 min to retrieve antigens. Then the sections were soaked in 3% hydrogen peroxide for 10 min to remove endogenous peroxidase. Sections were incubated with sheep serum at room temperature for 1 h to block nonspecific binding and then incubated at 4 ℃ overnight with the following primary antibodies: Rabbit anti-T-bet antibody (Proteintech, 13,700-1-AP, 1:400, USA); Mouse anti-Gata3 antibody (abcam, ab282110, 1:2000, UK); Rabbit anti-RORγt antibody (Proteintech, 13,205-1-AP, 1: 200, USA); Rabbit anti-Foxp3 antibody (Novus, NB100-39002, 1:400, USA). Following incubation with horseradish peroxidase (HRP) conjugated secondary antibody (DD13: ivision™ Poly-HRP sheep anti-mouse/rabbit secondary antibody, TALENT, China) at room temperature for 30 min, the 3,3-diaminobenzidine tetrahydrochloride substrate chromogen solution was used to detect signals and hematoxylin was used to redye the nucleus. The images were captured and then analyzed by Image J.
3
Immunofluorescence Detection of HIF-1α and FoxP3
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Tissue slices
on slides were permeabilized in PBS containing 0.1% Triton X-100 (PBST)
for 30 min, blocked with 1% serum, and then stained with 1:100 diluted
antibodies HIF-1α (NB100–134SS, Novus Biological, Littleton,
CO) and FoxP3 (NB100–39002, Novus Biological, Littleton, CO).
The samples were washed in PBST three times and then incubated with
FITC-tagged goat antirabbit secondary antibody (Novus Biological,
Littleton, CO) for 1 h at room temperature. The histology slides were
counterstained with Hoescht 33258 dye for 15 min, washed three times
with PBST, and analyzed with fluorescent microscopy. ImageJ version
1.53t was used to quantify the fluorescence intensity.
on slides were permeabilized in PBS containing 0.1% Triton X-100 (PBST)
for 30 min, blocked with 1% serum, and then stained with 1:100 diluted
antibodies HIF-1α (NB100–134SS, Novus Biological, Littleton,
CO) and FoxP3 (NB100–39002, Novus Biological, Littleton, CO).
The samples were washed in PBST three times and then incubated with
FITC-tagged goat antirabbit secondary antibody (Novus Biological,
Littleton, CO) for 1 h at room temperature. The histology slides were
counterstained with Hoescht 33258 dye for 15 min, washed three times
with PBST, and analyzed with fluorescent microscopy. ImageJ version
1.53t was used to quantify the fluorescence intensity.
4
Immunohistochemical Profiling of T-Cell Subsets
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Immunohistochemical staining was performed for T-bet (1:500; sc-21763; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) to evaluate Th1 cells; GATA binding protein 3 (GATA3; 1:500; sc-268; Santa Cruz Biotechnology, Inc.) was used for the Th2 cells; forkhead box protein P3 (FOXP3; 1:200; NB100-39002; Novus Biologicals, Centennial, CO, USA) was used for regulatory T (Treg) cells; and RAR-related orphan receptor (ROR)γ (1:1000; ab207082; Abcam, Cambridge, UK) was used for Th17 cells.
The sections were rehydrated, subjected to antigen retrieval using Dako Target Retrieval Solution (pH 9.0; Dako North America, Inc., Carpinteria, CA, USA) for 10 min at 121 °C, blocked for endogenous peroxidase with 0.3% H2O2 in methanol, and incubated for 30 min. After washing with phosphate-buffered saline, the sections were blocked for nonspecific binding using Blocking One Histo (Nacalai Tesque, Inc., Kyoto, Japan) for 15 min at room temperature and then incubated with the primary antibody overnight at 4 °C. The sections were reacted using peroxidase stain 3,3-diaminobenzidine (DAB) kit (Nacalai Tesque, Inc.) for 1 h and developed with DAB solution. Hematoxylin counterstaining was performed following the DAB reaction.
The sections were rehydrated, subjected to antigen retrieval using Dako Target Retrieval Solution (pH 9.0; Dako North America, Inc., Carpinteria, CA, USA) for 10 min at 121 °C, blocked for endogenous peroxidase with 0.3% H2O2 in methanol, and incubated for 30 min. After washing with phosphate-buffered saline, the sections were blocked for nonspecific binding using Blocking One Histo (Nacalai Tesque, Inc., Kyoto, Japan) for 15 min at room temperature and then incubated with the primary antibody overnight at 4 °C. The sections were reacted using peroxidase stain 3,3-diaminobenzidine (DAB) kit (Nacalai Tesque, Inc.) for 1 h and developed with DAB solution. Hematoxylin counterstaining was performed following the DAB reaction.
5
Immunohistochemistry Analysis of Tumor Immune Markers
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Immediately after harvest, a portion of the tumor tissue was fixed in 4% paraformaldehyde. Immunohistochemistry was performed on fixed tissues as previously described in detail [28 (link)]. The following primary antibodies were used: anti-CD4 (ab183685, Abcam, Cambridge, UK), anti-CD8 (ab203035, Abcam), anti-FOXP3 (NB100-39002, Novus Biologicals, Littleton, CO, USA), and anti-PD-L1 (ab2025921, Abcam). Images of stained slides were collected at a magnification of 40× using an Axioskop 40 light microscope (Carl Zeiss, Jena, Germany) and AxioVision 4.7 software. Optical density was quantified using ImageJ software ver. 1.8 (National Institutes of Health, Bethesda, MD, USA). The mean density of three sections per sample was calculated.
6
Immunofluorescence Analysis of Immune Markers
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The frozen sections were rinsed with Tris-buffered saline (TBS) at room temperature for 10 min. After blocking with 10% normal goat serum (NGS) containing blocking solution (10% NGS/TBS) for 1 h, each antibody was reacted in the humid chamber at 4 °C overnight. The concentration of each antibody used in the experiments was 1:500 for CD86 (bs-1035R: Bioss, Boston, MA, USA), 1:1000 for Foxp3 (NB100-39002: Novus Biologicals, Centennial, CO, USA) and 1:500 for Axin2 (ab32197: Abcam, Cambridge, UK). After the reaction with these primary antibodies, the bound antibodies were visualized with a VECTASTAIN Elite ABC kit (VECTOR LABORATORIES, Burlingame, CA, USA) and Diaminobenzidine (DAB) (ImmPACT DAB: VECTOR LABORATORIES). The tissues were counter stained with Mayer’s hematoxylin solution, and then dehydrated, penetrated, and sealed with MOUNT-QUICK.
For immunofluorescence analysis, the sections were sequentially reacted with the anti-CD68 antibody (1:500, ab125212: Abcam) and then reacted with the biotinylated secondary antibody. After the reactions, the sections were reacted with APC-streptavidin (405,207: BioLegend, San Diego, CA, USA) and the FITC-anti-CD206 antibody (1:200: bs-4747R-FITC, Bioss). Finally, the sections were stained with DAPI (Sigma) and observed under a fluorescence microscope (BZ-X810: KEYENCE).
For immunofluorescence analysis, the sections were sequentially reacted with the anti-CD68 antibody (1:500, ab125212: Abcam) and then reacted with the biotinylated secondary antibody. After the reactions, the sections were reacted with APC-streptavidin (405,207: BioLegend, San Diego, CA, USA) and the FITC-anti-CD206 antibody (1:200: bs-4747R-FITC, Bioss). Finally, the sections were stained with DAPI (Sigma) and observed under a fluorescence microscope (BZ-X810: KEYENCE).
7
Foxp3+ T Cell Quantification in Bovine Intestine
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Immunohistochemical analysis was carried out in a total of 20 jejunal tissue sections (with lymphoid tissue) and other 20 jejunal lymph node samples, one from each animal finally included in the study. As stated, these sections were representative of the lesion category assigned to each animal. Tissue sections 3-μm thick were mounted on electro charged adhesive gelatin-coated microscope slides (Thermo Scientific, Waltham, MA, USA). For the detection of Foxp3+ T lymphocytes, a primary polyclonal antibody (rabbit IgG isotype) against bovine Foxp3 (NB100-39002; Novus Biologicals®, Centennial, USA), at a 1:150 dilution, was used. The trading house had reported through a verified customer review that this Foxp3 antibody showed reactivity in sections of formalin-fixed bovine tissues (unpublished data). Heat-mediated antigen retrieval was performed by means of PT Link® system, using pH 6.0 target retrieval solution (Dako-Agilent® technologies, Santa Clara, USA) for 20 min at 95°C. Immunohistochemical procedure was carried out as described elsewhere [16 (link)]. Appropriate species-and isotype-matched immunoglobulins were used as control. These included sections with an isotype control for the primary antibody, and the omission of the primary antibody.
8
Evaluating T Cell Subsets by IHC
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Immunohistochemical staining was performed for T-bet (1:500; sc-21763, SANTA CRUZ BIOTECHNOLOGY, INC., Dallas, TX, USA) to evaluate the Th1 cells, GATA3 (1:500; sc-268, SANTA CRUZ BIOTECHNOLOGY, INC., Dallas, TX, USA) to evaluate Th2 cells, Foxp3 (1:200; NB100-39002, Novus Biologicals, Centennial, CO, USA) to evaluate Treg cells, and RORγ (1:1000; ab207082, Abcam, Cambridge, UK) to evaluate Th17 cells. The sections were rehydrated, subjected to antigen retrieval using Dako Target Retrieval Solution (pH 9.0; Dako North America Inc., Carpinteria, CA, USA) for 10 min at 121 °C, blocked for endogenous peroxidase with 0.3% H2O2 in methanol, and incubated for 30 min. After washing with PBS, the sections were blocked for non-specific binding using Blocking One Histo (Nakalai Tesque, Inc., Kyoto, Japan) for 15 min at room temperature and then incubated with the primary antibody overnight at 4 °C. Subsequently, the sections were reacted with Peroxidase Stain DAB Kit (Nakalai Tesque, Inc., Kyoto, Japan) for 1 h and developed with 3,3′-diaminobenzidine (DAB) solution. Hematoxylin counterstaining was performed following the DAB reaction.
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