Coronal cryosections (12 μm) of rat brains were obtained from the center of the anterior-posterior extent of the tumor. Tissues were blocked for 1 h in 2% donkey serum with 0.2% Triton X-100 and labeled overnight at 4°C with primary antibodies against GFAP (1:500; C9205, Sigma-Aldrich), ED-1 (1:200; MAB 1435, Millipore, Billerica, MA), RECA-1 (1:200; MA1-81510, Thermo Fisher Scientific Inc.), HIF-1 (1:200; sc-12542, Santa Cruz Biotechnology, Inc.), CD44 (1:200; ab157107, Abcam), Olig2 (1:100; ab109186, Abcam), CD3 (1:200; ab5690, Abcam), CD8a (1:200; ab33786, Abcam), CD4 (1:200; sc-1573, Santa Cruz Biotechnology, Inc, CD49d (1:200; ab22858, Abcam), P2Y12 (1:200; AS-55043, AnaSpec, Fremont, CA) or IBA1 (1:100; ab5076, Abcam). Alexa Fluor 500- or fluorescein isothiocyanate-conjugated secondary antibodies were applied, and tissues were coverslipped with ProLong Gold antifade reagent (P36930; Thermo Fisher Scientific Inc.). Specific labeling was confirmed by omission of primary antibody. Immunolabeled tissues were visualized with epifluorescence microscopy (Nikon Eclipse 90i; Nikon Instruments Inc., Melville, NY).
Ab33786
Manufactured by Abcam
Sourced in United Kingdom
Ab33786 is a laboratory product offered by Abcam. It is a type of lab equipment used for scientific research purposes. The core function of this product is to [CORE FUNCTION]. No further details on the intended use of this product can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab16669, by Abcam (2 mentions)
Ab215206, by Abcam (2 mentions)
Ab237722, by Abcam (2 mentions)
Ab4059, by Abcam (2 mentions)
Ab157107, by Abcam (1 mentions)
Ab109186, by Abcam (1 mentions)
Ab22858, by Abcam (1 mentions)
Ab5076, by Abcam (1 mentions)
Mab1435, by Merck Group (1 mentions)
Ab5690, by Abcam (1 mentions)
Ma1 81510, by Thermo Fisher Scientific (1 mentions)
C9205, by Merck Group (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Eclipse 90i, by Nikon (1 mentions)
Ab87099, by Abcam (1 mentions)
Ab178945, by Abcam (1 mentions)
Hoechst reagent, by Beyotime (1 mentions)
Ab65856, by Abcam (1 mentions)
Mab377, by Merck Group (1 mentions)
Af 212 na, by R&D Systems (1 mentions)
14 protocols using ab33786
1
Immunohistochemical Analysis of Rat Brain Tumor Microenvironment
2
Immunofluorescence Detection of Immune Cells
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The changes in infiltration levels of 21 immune cells were evaluated through the CIBERSORT algorithm, which quantifies the cell composition of sample tissue based on the gene expression profiles (21 (link)).
To verify the immunogenic effects of NOS2, we performed immunofluorescence detections on two pairs of clinical samples with different NOS2 protein levels. Immunofluorescence was conducted as previously described (22 (link)). Briefly, 4-mm tissue sections of HB clinical samples were prepared, and dewaxed with xylene and anhydrous ethanol. Then, the tissue sections underwent antigen repair, fixation, and nonspecific blocking. Next, the sections were incubated with first and secondary antibodies (NOS2, ab178945; CD8, ab33786; CD163, ab87099; Abcam, Cambridge, UK) in conditions protected from light. The slides were then sealed with anti-fluorescence quenching sealing tablets. CD8 and CD163 were stained with the Cy3-labeled goat anti-mice IgG (H&L) antibody (P0193; Beyotime, Shanghai, China). NOS2 was stained with the FITC-labeled goat anti-rabbit IgG (H&L) antibody (P0186; Beyotime). Nuclei were stained with Hoechst reagent (C0003; Beyotime). Subsequently, the slides were analyzed with an automatic fluorescent microscope using a 40× objective lens (BX53; Olympus, Tokyo, Japan).
To verify the immunogenic effects of NOS2, we performed immunofluorescence detections on two pairs of clinical samples with different NOS2 protein levels. Immunofluorescence was conducted as previously described (22 (link)). Briefly, 4-mm tissue sections of HB clinical samples were prepared, and dewaxed with xylene and anhydrous ethanol. Then, the tissue sections underwent antigen repair, fixation, and nonspecific blocking. Next, the sections were incubated with first and secondary antibodies (NOS2, ab178945; CD8, ab33786; CD163, ab87099; Abcam, Cambridge, UK) in conditions protected from light. The slides were then sealed with anti-fluorescence quenching sealing tablets. CD8 and CD163 were stained with the Cy3-labeled goat anti-mice IgG (H&L) antibody (P0193; Beyotime, Shanghai, China). NOS2 was stained with the FITC-labeled goat anti-rabbit IgG (H&L) antibody (P0186; Beyotime). Nuclei were stained with Hoechst reagent (C0003; Beyotime). Subsequently, the slides were analyzed with an automatic fluorescent microscope using a 40× objective lens (BX53; Olympus, Tokyo, Japan).
3
Immunohistochemical Staining Protocol
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The animals were perfused and processed for histological analysis as described previously.9 (link) Immunohistochemistry was performed as described previously.10 (link) The following primary antibodies were used: rabbit anti-pRPS6 (#2211, 1:400, Cell Signaling Technology, Danvers, MA, USA), goat anti-human GDNF (AF-212-NA, 1:1,000, R&D Systems Europe), rabbit anti-DHFR DD (1:20,000, a kind gift from the Wandless lab that has been used previously21 (link)), mouse anti-RFP (ab65856, 1:1,000, Abcam, Cambridge, UK), mouse anti-NeuN (MAB377, 1:1,000, Merck Millipore, Darmstadt, Germany), rat anti-CD11B (MCA275R, 1:1,000, Bio-Rad Laboratories, Solna, Sweden), and mouse anti-CD8 alpha (ab33786, 1:1,000, Abcam, Cambridge, UK). The secondary antibodies used were as follows: biotinylated horse anti-mouse (BA2001, 1:200, Vector Laboratories, Peterborough, UK), biotinylated horse anti-goat (BA9500, 1:200, Vector Labs), and biotinylated goat anti-rabbit-biotin (BA1000, 1:200, Vector Labs).
4
Immunofluorescence Staining of T-Cell Subsets
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Paraffin sections were de-paraffinized, rehydrated and antigen retrieval according to standard protocols. Then, slices were incubated with anti-CD3 (1:200, Abcam, ab16669), anti-CD4 (1:200, Abcam, ab237722), anti-CD8 (1:200, Abcam, ab33786), and anti-Foxp3 (1:100, Abcam, ab215206). Fluorophore-conjugated secondary antibodies were incubated for one hour at room temperature (1:200, Abcam). The slides were imaged with fluorescence microscopes (Leica, Barcelona, Spain).
5
Immunohistochemical Analysis of Heart Tissue
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Hearts were excised and fixed in zinc formalin at harvest (five months post-RT) or at time of death from those that died from heart failure due to treatment prior to the five-month time point. Tissues were fixed for 48 h and then transferred to 70% ethanol. Tissue processing and immunohistochemical analyses were reported previously [26 (link)]. For T-cell staining, anti-CD3 (ab16669, Abcam, Cambridge, UK) and anti-CD8α (ab33786, Abcam, Cambridge, UK) were used to detect immune cell populations on heart tissue at 10–11 weeks post-RT. All images were acquired using a Nikon Eclipse 50i upright microscope equipped with a Nikon Digital sight DS-U3 camera and NIS Elements BR software (Nikon Instruments, Melville, NY, USA). CD3 and CD8α T-cell staining was quantified by an automated count to detect the number of cells per high-power field above the set threshold or counted by counting the number of cells per high-power field on blinded samples as previously reported, with the observer blinded to the experimental conditions [26 (link)].
6
Immunohistochemical Staining for CD3 and CD8
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Formalin-fixed and paraffin-embedded tissue sections were deparaffinized in xylene and graded alcohols, hydrated and washed in PBS. After antigen retrieval in a sodium citrate buffer (pH 6) in a microwave oven, the endogenous peroxidase was blocked by 0.3% H2O2 for 15 min. Sections were incubated overnight at 4 °C with primary antibody, rabbit polyclonal anti-CD3 (clone A0452, Dako) or mouse monoclonal anti-CD8 (ab33786, Abcam). As a secondary antibody, the anti-rabbit-horseradish peroxidase (HRP) SuperPicTure Polymer detection kit (Invitrogen) or Envision system HRP-anti-mouse (Dako) was used. A matched isotype control was used as a control for nonspecific background staining.
7
Comprehensive Cardiac Cell Characterization
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Antibodies against Ki67 (ab16667, Abcam, 1:200), α-Sarcomeric Actinin (SA, ab9465, Abcam, 1:200), vWF (ab6994, Abcam, 1:100), CD31 (ab28364, Abcam, 1:100), Podoplanin (ab10288, Abcam, 1:500), Vimentin (ab92547, Abcam, 1:500), Sca-1 (ab109211, Abcam, 1:200), α-SMA (ab32575, Abcam, 1:500), MPO (PA5-16672, Thermo Fisher, 1:100), CD4 (ab237722, Abcam, 1:200), CD8 (ab33786, Abcam, 1:100), cTnT (MS-295P, Invitrogen, 1:100), Nkx2.5 (ab106923, Abcam, 1:100) as well as Alexa Fluo 594 or 488 conjugated Goat anti Rabbit or mouse secondary antibodies (1:500) were purchased from Abcam. TUNEL staining kit was purchased from Promega (G3250).
8
Immunohistochemical Profiling of Tumor Microenvironment
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Prepared TMAs were put into xylene for deparaffinization and into gradient alcohol for rehydration, followed by antigen retrieval with citrate buffer. Then, their endogenous peroxidases were inactivated in 0.3% H2O2 at 37℃ for 20 minutes. After blocking in 10% bull serum albumin (BSA) for 1 hour, TMA was incubated with indicated primary antibodies at 4℃ overnight. Primary antibodies contain anti‐DKK4 (1:200 Abcam, ab38589), anti‐CD8A (1:400 Abcam, ab33786), and anti‐β‐catenin (1:500, Abcam, ab32572) antibodies. After combining with special HRP‐conjugated secondary antibody, the local proteins were marked with DAB substrate kit (Cell Signaling Technology) and the images were observed and captured by Nikon microscope. The profiles of local expression were described by four levels: negative as 0‐score, weak as 1, moderate as 2 and positive as 3, stratified by the staining intensity and positive area rates. Anti‐CD8A antibody was used for mark CD8+ T cells, and the mottling positive region combined with a miner nucleus was counted as one cell.
9
Immunohistochemical Analysis of Tumor Microenvironment
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Formalin‐fixed paraffin‐embedded tissues from the CT26 tumor model and AOM‐DSS mice were sectioned at 10 μm thickness. After blocking, the sections were incubated with specific primary antibodies followed by incubation with secondary antibodies conjugated with horseradish peroxidase or fluorescent dye. All immunohistochemistry images were acquired using the OLYMPUS CellSens Standard 1.18 system. The primary antibodies used in immunohistochemistry were as follows: CD8 (1: 200 dilution, ab33786, Abcam, Cambridge, UK), Granzyme B (1:100 dilution, ab4059, Abcam), PD‐1 (1:100 dilution, GB13338, Servicebio, Hubei, China), CD11b (1:100 dilution, Ab133357, Abcam), Gr1 (1:50 dilution, Mab1037, R&D system, Minneapolis, MN, USA), CD206(1:100 dilution, ab64693, Abcam), p‐CREB (1:200 dilution, S113, ab32096, Abcam), p‐Stat3 (1:100 dilution, Tyr705, 9145, CST, Danvers, MA), p‐Akt (1:100 dilution, Ser473, 4060, CST), and Arg‐1 (1:200 dilution, 16001, Proteintech, Rosemont, IL, USA).
10
Quantifying CD8+ T Cell Infiltration in Mammary Tumors
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To assess CD8+ infiltration in mammary tumors, IHC assays were performed. Five micrometer paraffin sections, cut transversely, were deparaffinized and rehydrated from xylene through a graded series of ethanol. Antigen retrieval was performed in a high-pH Target Retrieval Solution (pH 9, DAKO S2368) in a microwave for 15 min, followed by 20 min of cooling at room temperature. Then, endogenous peroxidases were blocked with 3% hydrogen peroxide in water (DAKO, K0679) and non-specific staining was blocked with protein block serum-free (DAKO, X0909). Immunostaining with CD8 (Abcam, ab33786, 1:100) was performed overnight following the manufacturer’s protocol for LSAB-HRP immunohistochemistry kit (DAKO, K0679). Slides were examined under a bright-field microscope (Olympus BX 61 with Qiacam scanning camera) and image software package CellSens (Waltham). Micrographs were taken at 20× magnification. Total number of positive cells per field was calculated.
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