The tissue is fixed in formalin and embedded in paraffin, and then cut into thin slices. The sections were deparaffinized with xylene and hydrated with ethanol, and then the antigen was recovered by pressure cooking. At room temperature, the sections were sliced with TNC primary antibody (ab108930, Abcam, Cambridge, MA), MMP9 (Abcam, ab76003, 1:1000 dilution), MMP2 (Abcam, ab92536, 1:1000 dilution) and CD31 (ab134168, Abcam), incubated for 60 min and then incubated with IgG H&L (HRP; 1:200 dilution, Abcam, Cambridge, UK). Then, the sections were stained with chromogen and counterstained with hematoxylin. The score is based on the intensity of staining (0 means no staining, 1 means weak staining, 2 means moderate staining, and 3 means strong staining). The product of the two levels was calculated as the final expression score.
Ab134168
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab134168 is a laboratory reagent. It is a monoclonal antibody that specifically binds to a target antigen. The core function of this product is to detect and quantify the target antigen in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab6721, by Abcam (3 mentions)
Ab108930, by Abcam (2 mentions)
Ab76003, by Abcam (2 mentions)
Ab92547, by Abcam (2 mentions)
Ab53465, by Abcam (2 mentions)
Ab92536, by Abcam (1 mentions)
Pa5 40772, by Thermo Fisher Scientific (1 mentions)
Ab28364, by Abcam (1 mentions)
Ar0009, by Boster Bio (1 mentions)
Vectra automated quantitative pathology imaging system, by PerkinElmer (1 mentions)
Inform software, by PerkinElmer (1 mentions)
Ab15580, by Abcam (1 mentions)
Ab81289, by Abcam (1 mentions)
Pa5 103744, by Thermo Fisher Scientific (1 mentions)
Aperio imagescope software, by Leica (1 mentions)
Ab157489, by Abcam (1 mentions)
H e c0105, by Beyotime (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Ab7213, by Abcam (1 mentions)
Alexa fluor 488 donkey anti mouse igg, by Antgene (1 mentions)
16 protocols using ab134168
1
Immunohistochemical Analysis of Tissue Samples
2
Immunohistochemical Analysis of Tumor Markers
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Formalin-fixed, and paraffin-embedded tissues were sectioned at 4 mm thickness, then harvested and fixed in 4% paraformaldehyde overnight at 4 oC. The antigen blocking was performed using 10% goat serum (AR0009,Boster,China). The sections were probed overnight at 4 oC with primary anti-Foxq1 (PA5-40772, Invitrogen, USA), anti-CD31 (ab134168, Abcam, USA) and anti-EGFR (ZM-0093, Zsbio, China) antibodies. The staining was detected using the DAB system (ZLI-9017, Zsbio, China). To detect VM structures, a PAS staining kit (G1281, Solarbio, China) and anti-CD31 (ab28364, Abcam, USA) were used. The numbers of positive cells were counted from ≥5 randomly chosen fields and at ×400 magnification, by two independent pathologists.
3
Multicolor Immunofluorescence Imaging of SLC25A38, CBP, and CD31
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OPAL-4-plex reagents (Perkin-Elmer) were used for multi-color IF according to instructions. Images were acquired on the Vectra Automated Quantitative Pathology Imaging System (Perkin-Elmer), and analyzed using Inform software (Perkin-Elmer) for the SLC25A38, CBP, and CD31 expression rate in the entire tissue sections. Anti-SLC25A38 (Abcam, ab133614), anti-CBP (Santa Cruz, sc-365387) and anti-CD31 (Abcam, ab134168) were used at dilutions of 1:100, 1:100 and 1:1000, respectively.
4
Immunohistochemical Analysis of CMTM7, CD34, CD31, and Ki-67
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Immunohistochemical staining was used to determine the expression of CMTM7, CD34, CD31, and Ki-67 in the tissues. Cancer tissues were fixed in 4% paraformaldehyde phosphate buffer for 12 h, dewaxed with xylene and hydrated with gradient alcohol (anhydrous ethanol, 95% ethanol and 75% ethanol for 3 min each). The tissues were heated in 0.01 M citrate buffer solution for 15–20 min, and cooled to room temperature, followed by washing with PBS. Thereafter, the tissues were probed with 30 μL primary antibodies against CMTM7 (PA5-103744, 1:200, Thermo Fisher), CD34 primary antibody (ab81289, 1:200, Abcam, Cambridge, UK), CD31 primary antibody (ab134168, 1:100, Abcam) and Ki-67 primary antibody (ab15580, 1:200,Abcam), and then re-probed with 30 μL of secondary antibody goat anti-rabbit IgG (ab6721, 1:2000, Abcam) for 1 h at room temperature. After PBS washing, streptavidin-peroxidase was added dropwise to the tissues and placed at 37 °C for 30 min, followed by DAB development for 5–10 min. Following tap water washing for 10 min, the tissues were counterstaining with hematoxylin for 2 min, differentiated with hydrochloric acid alcohol, dehydrated, cleared and mounted before microscopic observation.
5
Immunohistochemical Analysis of LRRC8A in Cancer Tissues
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Tissue microarrays were commercial and purchased from the Shanghai Outdo Biotech Co., Ltd. (Shanghai, China) (HDgS-C120PT-01, HOrg-C120PG-02, HDgS-C140PT-01, HRec-Ade180Sur-01 and HCol-Ade180Sur-02); ethics approval for research using human tissue was obtained from the Ethics Committee in Taizhou Hospital, Zhejiang Province, and included a waiver for consent. Patients involved in HRec-Ade180Sur-01 underwent surgery during the period from July 2006 to August 2007, and then were followed up in August 2013. Patients involved in HCol-Ade180Sur-02 underwent surgery during the period from May 2007 to April 2008, and were followed up in September 2014. The tissues or tissue microarrays were immunolabeled using the UltraSensitive™ SP system (KIT-9710; Fuzhou Maixin Biotech Co., Ltd., Fuzhou, China) and stained with hematoxylin and eosin (H&E; C0105; Beyotime Institute of Biotechnology, Haimen, China) following the manufacturer's instructions; the dilution of primary antibodies for LRRC8A (cat. no. ab157489; Abcam, Cambridge, MA, USA) and CD31 (cat. no. ab134168; Abcam) was 1:100. Images were captured under the digital pathology whole slide scanners. Using Aperio ImageScope software (Leica Microsystems, Inc., Buffalo Grove, IL, USA), parenchyma in cancer and cancer adjacent tissues were depicted, and the intensity and distribution of the immunostaining reaction (LRRC8A) were analyzed.
6
Immunohistochemical and Immunofluorescence Analysis of IL-15 Expression
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Paraffin sections of formalin‐fixed samples were cut into 5 um thick sections and analyzed using an immunohistochemistry kit (Neobioscience, Wuhan, China). All sections were first incubated with IL-15 primary antibody at a 1:100 dilution (ab7213, Abcam, Cambridge, UK) at 4 °C overnight. The sections were then incubated with reagent B from the kit and finally, sections were stained with DAB working reagent for 2 to10 min depending on the degree of stain required and the cell nucleus stained with hematoxylin.
For immunofluorescence, all sections were first incubated with IL-15 primary antibody (ab7213, Abcam, Cambridge, UK) in combination with CK7 (YM3054, Immunoway, Beijing, China) or Vimentin (ab92547, Abcam, Cambridge, UK) or CD31 (1:150, ab134168, Abcam, UK) at 4 °C overnight. Then, the sections were incubated with a secondary antibody, Alexa Fluor 594 Donkey Anti rabbit IgG or Alexa Fluor 488 Donkey Anti mouse IgG (Antgene, Wuhan, China). The DNA dye-4′, 6-diamidino-2-phenylindole (DAPI), was used for nucleus staining. In the control experiments, primary antibodies were omitted. Images were captured using a fluorescence microscope (Olympus BX53, Tokyo, Japan) and the mean values of area-integrated optical densities analyzed using Image-Pro Plus 6.0 (Media Cybernetics, Denver, USA).
For immunofluorescence, all sections were first incubated with IL-15 primary antibody (ab7213, Abcam, Cambridge, UK) in combination with CK7 (YM3054, Immunoway, Beijing, China) or Vimentin (ab92547, Abcam, Cambridge, UK) or CD31 (1:150, ab134168, Abcam, UK) at 4 °C overnight. Then, the sections were incubated with a secondary antibody, Alexa Fluor 594 Donkey Anti rabbit IgG or Alexa Fluor 488 Donkey Anti mouse IgG (Antgene, Wuhan, China). The DNA dye-4′, 6-diamidino-2-phenylindole (DAPI), was used for nucleus staining. In the control experiments, primary antibodies were omitted. Images were captured using a fluorescence microscope (Olympus BX53, Tokyo, Japan) and the mean values of area-integrated optical densities analyzed using Image-Pro Plus 6.0 (Media Cybernetics, Denver, USA).
7
Immunohistochemical Analysis of Ocular Tumors
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Each FFPE eye was cut into multiple 4-μm sections in the area of the central tumor cross-section. One section per tumor was then stained with PAS without hematoxylin counterstain. The other four sections were pretreated in EDTA buffer at pH 9.0 for 20 min, and incubated with mouse monoclonal antibodies against BAP-1 (sc-28383; Santa Cruz Biotechnology, Dallas, TX) at dilution 1:40, against CD31 (ab134168; Abcam PLC, Cambridge, UK) at dilution 1:400, against laminin (m063801–2; Dako, Agilent Technologies, Santa Clara, CA) at dilution 1:20, and against CD68 (M087601–2, Dako) at dilution 1:200, according to the manufacturers’ instructions. A red chromogen was used. Finally, the slides were counterstained with hematoxylin and rinsed with deionized water. The deparaffinization, pretreatment, primary staining, secondary staining and counterstaining steps were run in a Bond III automated immunohistochemistry (IHC) and in situ hybridization (ISH) stainer (Leica, Wetzlar, Germany). The dilutions were gradually titrated until optimal staining was achieved, according to manual control. All slides were then digitally scanned at 40X, using a Nano Zoomer 2.0 HT (Hamamatsu Photonics K.K., Hamamatsu, Japan) at the Winship Research Pathology Core Laboratory, Winship Cancer institute of Emory University (Atlanta, GA).
8
HEK293T, KYSE30, and OE19 Cell Hypoxia
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The cell lines HEK293T were got from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cell lines KYSE30 and OE19 were obtained from fenghbio (Hunan, China). Antibodies for ATF5 (ab184923), VEGFA (ab52917), TIMP1 (ab61224), TWIST1 (ab175430), EGF (ab206423), CD31 (ab134168) and POL II (ab264350) were bought from Abcam (Cambridge, MA, USA). Antibodies for PDK1 (13037), PGK1 (68540), CA9 (5649), CBP (7389), P300 (54062), HIF1α (14179), HIF1β (5537) and flag-tag (2368) were procured from Cell Signaling Technology. The human VEGFA ELISA Kit (EK0539) was purchased from BOSTER Biological Technology (Wuhan, China). Identified primers were synthesized by TSINGKE Biological Technology (Beijing, China). For hypoxic conditions, cells were cultured in a sealed hypoxia chamber with a mixture of 1% O2, 5% CO2, and 94% N2 for 2 h.
9
Immunofluorescence Staining of Cellular Markers
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Cells were seeded onto sterile cover slips in a Corning 12-well culture plate at density of 104 cells/mL and treated according to the experimental design. At the indicated time point, cells were washed three times with PBS for 10 min each, then fixed with 4% paraformaldehyde at 37 °C for 15 min in a thermostatic water bath, washed with PBS for 10 min each, and then permeabilized using 0.4% Triton X-100 for 30 min at 37 °C. After cells were blocked with goat serum for 30 min, cells were incubated with the primary anti-CK19 (ab52625, Abcam, Cambridge, MA, USA), anti-vimentin (ab193555, Abcam, Cambridge, MA, USA), anti-CD31 (ab134168, Abcam, Cambridge, MA, USA), VEGF (ab32152, Abcam, Cambridge, MA, USA), and anti-vWF (ab6994, Abcam, Cambridge, MA, USA) antibodies overnight, followed by incubation with the corresponding fluorophore-conjugated antibodies for 60 min, then cells were washed with PBST for 10 min each and stained with DAPI for 5 min. The cover slips were carefully removed and then mounted on slides with glycerol. The same protocol was performed in the negative control groups except that the primary antibodies were omitted. The slides were observed by confocal microscopy (DFM-80C, Nikon, Japan), and images were assessed by Nikon auxiliary systems. The results of immunofluorescence were quantified using the Image Pro Plus software.
10
Immunohistochemical Analysis of Glioma Proteins
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IHC staining was performed as reported previously35 (link). The expression level of TNC in glioma tissues was scored as the proportion of the area with positive staining (0–100%) multiplied by the staining intensity (0, negative; 1, weak; 2, moderate; 3, intense). The scores were determined by two pathologists independently. The median score was chosen as the cut-off value for defining high and low expression. Sections were probed with primary anti-TNC (ab108930, Abcam, Cambridge, MA), anti-CD31 (ab134168, Abcam), anti-pAkt (4060P, Cell Signaling Technologies [CST], Danvers, MA,), anti-matrix metalloproteinase (MMP) 2 (ab86607, CST), and anti-MMP9 (ab76003, Abcam) antibodies overnight at 4 °C, and the antibodies were detected using the DAB system (Golden Bridge, Beijing, China).
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