Tumors were collected and fixed in 4% formalin. Three-micrometer-thick sections from paraffin-embedded tissues were mounted on glass slides, rehydrated and antigen retrieval was performed at 100°C (TRIS-Borate-EDTA (TBE), pH 8). Primary antibodies were used according to manufacturer’s instructions (Abcam, dilutions 1:200): anti-CD3 (SP7), anti-CD4 (EPR19514), anti-CD8 (ab203035). Slides were incubated with primary antibodies in moist chambers overnight at 4°C. Slides were washed with PBS before incubation with label-secondary goat antirabbit IgG for 1 hour at 4°C, washed three times, incubated with DAB substrate KIT (Abcam) solution for 10 min and washed with PBS. Background staining was performed with Mayer’s hematoxylin. Sections were dehydrated through ascending alcohols-to-xylene and mounted.
Ab203035
Manufactured by Abcam
Sourced in United Kingdom
Ab203035 is a type of lab equipment used for scientific research purposes. It is designed to perform specific functions within the laboratory setting. The core function of this product is to assist researchers in their experimental procedures, though the exact nature of its intended use is not provided in the details available.
Automatically generated - may contain errors
Lab products found in correlation
Ab183685, by Abcam (9 mentions)
Ab5690, by Abcam (3 mentions)
Ab54501, by Abcam (3 mentions)
Ab125212, by Abcam (3 mentions)
Ab64693, by Abcam (2 mentions)
Ab 214214, by Abcam (2 mentions)
Ab10558, by Abcam (2 mentions)
Ab182422, by Abcam (2 mentions)
Ab28364, by Abcam (2 mentions)
Dab substrate kit, by Abcam (1 mentions)
Peroxidase blocking reagent, by Agilent Technologies (1 mentions)
Ab33775, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab25333, by Abcam (1 mentions)
Ab91279, by Abcam (1 mentions)
Foxp3 clone 236a e7, by Abcam (1 mentions)
Clone epr19514, by Abcam (1 mentions)
Elivision super hrp mouse rabbit ihc kit, by Maixin Group (1 mentions)
Ab15323, by Abcam (1 mentions)
Sc 56, by Santa Cruz Biotechnology (1 mentions)
24 protocols using ab203035
1
Immunohistochemical Analysis of T-Cell Markers
2
Intestinal T-cell Immunohistochemistry
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The expressions of CD4+ and CD8+ T-cells in the intestinal mucosa were detected by immunohistochemistry. Sections of 5 μm thickness from paraffin-embedded rat intestine tissue blocks were prepared. The slides were initially deparaffinized, and, to each section, Peroxidase-Blocking Reagent (Agilent, Santa Clara, CA, USA) was added to block the endogenous peroxidase (10 min). The sections were incubated for 2 h with diluted primary Anti-CD4 antibody (1:100; ab33775; Abcam, Cambridge, UK) and Anti-CD8 antibody (1:100; ab203035; Abcam, Cambridge, UK). After incubation, the slides were washed with Tris-buffer-saline buffer and incubated with a secondary antibody for 30 min. Next, freshly prepared 3,3′-diaminobenzidine (DAB) was added (15 min). Slides were additionally stained with hematoxylin counterstain for 15 min, rinsed, and dehydrated in 75% and 100% absolute alcohol, and xylene. After gently placing a coverslip over the slides, the sections were examined microscopically for the presence of CD4+ and CD8+ T-lymphocytes. Further, quantitative analysis was performed on each CD4+T and CD8+T-lymphocyte stained section by counting positive cells manually in 20 random fields at a magnification of 400× on a computer monitor connected to an optical microscope (Olympus Optical BX51TF, Olympus, Tokyo, Japan).
3
Histological Analysis of Tumour Samples
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The H22 and 4T1 tumours harvested from PBS control and CD151 peptide-immunised groups were fixed with 10% formalin overnight and processed into 5-μm-thick paraffin sections. The slides were stained with haematoxylin and eosin (HE) or subjected to IHC with anti-mouse CD8 (Abcam, Cat #ab203035, UK), followed by HRP secondary antibody (Abcam, Cat #ab205718, UK) and DAB treatment. Images were obtained under a microscope (BX43, Olympus, Japan) at a magnification of 200×.
4
Immunohistochemical Analysis of Tumor Samples
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Irradiated cells were embedded in low-melting agarose followed by dehydration and paraffin embedding. Tumors from treated mice were collected, fixed in 4% formalin for 24 h at 4°C, immersed in 75% alcohol, and embedded in paraffin. Primary antibodies used for IHC were as follows: anti-PD-L1 (clone EPR20529; Abcam, Cambridge, UK), anti-PD-1 (clone EPR20665; Abcam), anti-forkhead box P3 (FOXP3; clone 236A/E7; Abcam), anti-CD4 (clone EPR19514; Abcam), anti-CD3 (ab5690; Abcam), and anti-CD8 (ab203035; Abcam), anti-Arginase (ab91279; Abcam), anti-iNOS (ab15323; Abcam), anti-NK1.1 (MA1-70100; Thermo Fisher), anti-MHC I (ER-HR52; Abcam), anti-MHC II (ab25333; Abcam), rabbit anti-mouse (550338; BD), rat anti-mouse (ab6733; Abcam), and mouse anti-mouse (553441; BD). IHC was carried out using an Elivision Super HRP (Mouse/Rabbit) IHC Kit (KIT-9922, Maixin Biotech) and a Catalyzed Signal Amplification System (K1500, Dako)，according to the manufacturer’s instructions. Three pathologists analyzed each tissue section. The percentages of positive cells in each visual field were assessed in high-power fields (magnification: 100×). Results were averaged and used for statistical analysis. The DLN and spleen were immunostained as positive controls (Supplementary Figure 2(c) ).
5
Immunohistochemical and Immunofluorescence Analyses of Tumor Samples
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Formalin-fixed and paraffin-embedded tumor samples were cut into 3.5-µm sections, deparaffinized, and antigen-retrieved using sodium citrate solution (0.01 M, PH: 6.0). Then, the sections were processed for antibody staining for immunohistochemistry (IHC) or immunofluorescence (IF). For IHC, slides were incubated with primary anti-PCNA (1:250, sc-56, Santa Cruz), anti-Foxp3 (1:100, 12,653, Cell Signaling Technology) or anti-CD8 Ab (1:200, ab203035, Abcam), and then secondary antibody HPR anti-mouse or rabbit IgG (Maxim). The slides were colored with 3, 3ʹ-Diaminobenzidine (DAB, Sigma-Aldrich) and counterstained by hematoxylin. For IF, anti-CD31 (1:250, sc-376,764, Santa Cruz), anti-CD206 (1:1000, ab64693, Abcam) and anti-F4/80 (1:250, sc-377,009, Santa Cruz) were used as primary Abs while anti-mouse IgG (H + L) (1:1000, 4408, Cell Signaling Technology) and anti-rabbit IgG (H + L) (1:1000, 4413, Cell Signaling Technology) were used as secondary Abs. The sections finally were mounted using DAPI-Fluoromount-G clear mounting agents (Southern Biotech, Birmingham, UK) and images were taken using fluorescence microscopy (Nikon, Japan). Positive cells were quantified using Image J software and expressed as the mean of the percentage of positive cells ± SD with 10 randomly selected fields per sample.
6
Immunohistochemical Analysis of IDO and T-cell Markers
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Paraffin-embedded specimens were prepared using standard methods. Sections were stained with hematoxylin and eosin (H & E) and immunohistochemical staining. The primary Abs were anti-IDO1 (ab106134; Abcam, Cambridge, UK), anti-IDO2 (ab214214; Abcam), anti-CD3 (ab5690; Abcam), anti-CD4 (ab183685; Abcam), anti-CD8 (ab203035; Abcam), anti-IL-17 (ab79056; Abcam), and anti-Ki-67 (NB500-170; Novus Biologicals, Centennial, USA). Histofine Simple Stain MAX PO (Nichirei Biosciences, Tokyo, Japan) was used for mouse samples and Dako Envision System-Labeled Polymer HRP (Dako, Tokyo, Japan) for human samples as secondary antibodies.
7
Immunohistochemical Analysis of Immune Cell Markers
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Paraffin sections were deparaffinized, hydrated and microwave-heated in either EDTA buffer (pH 8) or citrate buffer (pH 6). The endogenous peroxidase activity was blocked with 3% H2O2 in methanol and non-specific labeling was blocked by 5% bovine serum albumin. Sections were incubated overnight at 4 °C with the following primary antibodies: anti-CD45 (ab10558, Abcam, 1:500), anti-CD4 (ab183685, Abcam, 1:2000), anti-CD8 (ab203035, Abcam, 1:1000), anti-FoxP3 (ab54501, Abcam, 1:4000), anti-EMBP (sc-33938, Santa Cruz, 1:500), anti-CD163 (ab182422, Abcam, 1:500). For secondary antibodies, HRP-conjugated anti-rabbit (Cat. No. P0448, Dako, diluted 1:500) and anti-goat (sc-2020, Santa Cruz, 1:500) were applied for 1 h at room temperature, followed by standard 3,3′-diaminobenzidine (Cat. No. K3468, Dako) staining procedure. Sections were counterstained with hematoxylin.
8
Immunofluorescence Staining of Tumor Sections
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Thin paraffin-embedded tumor sections were subjected to antigen retrieval using a Diva Decloaker kit (Biocare Medical, Pacheco, USA), following the manufacturer’s instructions. Subsequently, samples were blocked with bovine serum albumin 3%, and the primary antibody was added (polyclonal rabbit anti-mouse natural cytotoxicity triggering receptor 1 [NCR1] or polyclonal rabbit anti-mouse CD8, both at 1:25 dilution; AB214468 and AB203035, respectively; Abcam). The antibodies were incubated at RT overnight. Thereafter, the slides were washed twice and incubated with the secondary antibody (goat anti-rabbit IgG-Alexa594; AB150080 at 1:2,000 dilution; Abcam) for 2 hours at RT. Finally, after washing, 3 µM DRAQ7 (DR70250; BioStatus, Leicestershire, UK) was added for 20 minutes at RT, and the slides were mounted using Fluoroshield (Sigma-Aldrich). Images were acquired under a confocal microscope (DM2500; Leica, Wetzlar, Germany) and analyzed with LASX software (Leica). Image analysis was carried out by scanning samples in different directions using 40 randomly selected images per sample.
9
Immunohistochemical Evaluation of CD8+ Lymphocytes in Tumor Samples
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Representatives of each experimental group were humanely euthanized once the tumors reached the experimental end point of 1000 mm3. Long-term survivors were euthanized at day 70 post-tumor implantation. Excision biopsies were preserved by formalin fixation and paraffin embedding for immunohistochemistry analysis (IHC). A histological evaluation was carried out at the Comparative Pathology Laboratory, Center of Comparative Medicine and Surgery at Icahn School of Medicine at Mount Sinai, New York (US). IHC staining of CD8+ lymphocytes was performed on 5-µm-thick tumor sections. A polyclonal, mouse-specific anti-CD8 antibody (Abcam, Cambridge, UK; ab203035) was used following the manufacturer’s instructions. The final counterstaining was performed using hematoxylin.
10
Immunohistochemical Evaluation of Gallbladder Tissue
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Formalin‐fixed paraffin‐embedded gallbladder sections (2 µm) were deparaffinized, rehydrated, and antigen‐retrieved by using pH 9.0 Tris/EDTA buffer, following antibody recommendations. Endogenous peroxidase activity was blocked for 15 min using 3% H2O2, and sections were incubated with indicated primary antibodies for 45 min at room temperature. Sections were then incubated with anti‐rabbit IgG secondary antibody for 30 min at room temperature (ImmPRESS™ Reagent; Vector Laboratories Inc., Burlingame, CA, USA). Next, sections were incubated with 33‐diaminobenzidine tetrachloride (DAB) and counterstained with hematoxylin. The following primary antibodies were used: F4/80 (ab111101, 1 : 100), CD3 (ab16669, 1 : 200), CD4 (ab183685, 1 : 1500), and CD8 (ab203035, 1 : 800), purchased from Abcam (Cambridge, UK). To determine the IHC score, a combined semiquantitative score was used for the assessment of immune cell infiltrates in FFPE sections, described elsewhere [17 ]. Two independent pathologists (X.G. and J.C.R) evaluated the following parameters: (a) abundance of inflammatory cells (0: absent; 1: low; 2: moderated; and 3: high) and (b) the intensity of the stain (0: absent; 1: weak; 2: moderated; and 3: strong). IHC score (from 0 to 9 values) was calculated by multiplication of these two parameters.
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