A tissue microarray (TMA) containing 756 primary ccRCC and corresponding normal tissue samples was used as described before [28] (link). For immunohistochemical staining of tissue, microarray slides or formalin fixed and paraffin-embedded tumor tissue antibodies directed against CD3 (IR503, Dako, Glostrup, Denmark), CD8 (C8/144B, Dako), CD20 (IR604, Dako), CD56 (123C3, Dako), CD68 (PG-M1, Dako), CD138 (MI15, Dako), FoxP3 (236A/E7, Abcam, Cambridge, United Kingdom), myeloperoxidase (MPO) (IR511, Dako), PD-1 (NAT105, Abcam), and PD-L1 (EPR19759, Abcam) were used. All slides were stained with automatized immunostainers (autostainer plus, Dako).
Pg m1
Manufactured by Agilent Technologies
Sourced in United States, Denmark, United Kingdom, Germany
The PG-M1 is a compact and precise microflow gradient pump from Agilent Technologies. Designed for a wide range of liquid chromatography applications, it provides accurate and reproducible solvent delivery with low pulsation. The PG-M1 features a compact design and straightforward user interface, making it a reliable and efficient choice for laboratory setups.
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Lab products found in correlation
Envision system, by Agilent Technologies (3 mentions)
Mib 1, by Agilent Technologies (2 mentions)
Biotinylated goat anti mouse igg, by Agilent Technologies (2 mentions)
Tp1020, by Leica (2 mentions)
Tissue studio 3, by Definiens (2 mentions)
Warp red, by Biocare Medical (2 mentions)
Mirax digital desk scanner, by Zeiss (2 mentions)
Ab64693, by Abcam (2 mentions)
Vectra automated quantitative pathology imaging system, by PerkinElmer (2 mentions)
Inform software, by PerkinElmer (2 mentions)
Rbt cd4, by Bio SB (2 mentions)
Epr4877, by Abcam (2 mentions)
Ventana discovery xt, by Roche (2 mentions)
Carb 3, by Agilent Technologies (2 mentions)
Cd206, by R&D Systems (2 mentions)
Epr1344, by Abcam (2 mentions)
Ep334, by ZSGB-BIO (2 mentions)
Leum1, by ZSGB-BIO (2 mentions)
Cd163, by ZSGB-BIO (2 mentions)
Cd169, by R&D Systems (2 mentions)
51 protocols using pg m1
1
Immunoprofiling of Renal Cell Carcinoma
2
Immunohistochemical Profiling of Brain Tumors
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Ten micrometre sections of formalin‐fixed paraffin‐embedded tissue from the tumour TMA blocks were immunostained in a single batch. Immunohistochemistry was performed using the appropriate antigen retrieval methods for each primary antibody. Biotinylated secondary antibodies (rabbit anti‐mouse, swine anti‐rabbit and rabbit anti‐goat) were from Dako, normal serum and avidin‐biotin complex were from Vector Laboratories. Bound antibody was visualized using the avidin‐biotin‐peroxidase complex method (Vectastain Elite ABC) with 3′3 diaminobenzidine as chromogen and 0.05% hydrogen peroxide as substrate to obtain a brown precipitate. All sections were dehydrated before to be mounted in DePeX (BDH Laboratory Supplies).
The primary antibodies used were: microglia: Iba1 (all microglia‐Wako Laboratories), CD68 (phagocytic activity – clone PG‐M1, Dako); stem cell: CD133 (orb18124, Biorbyt), nestin (ab22035, Abcam), SOX2 (clone Y‐17, Santa Cruz Biotechnology); GFAP (glial acid fibrillary protein – Z0334, Dako), and Ki67 (cell proliferation – clone MIB1, Dako).
The primary antibodies used were: microglia: Iba1 (all microglia‐Wako Laboratories), CD68 (phagocytic activity – clone PG‐M1, Dako); stem cell: CD133 (orb18124, Biorbyt), nestin (ab22035, Abcam), SOX2 (clone Y‐17, Santa Cruz Biotechnology); GFAP (glial acid fibrillary protein – Z0334, Dako), and Ki67 (cell proliferation – clone MIB1, Dako).
3
Breast Adipocyte Size and Metabolic Health
4
Immunohistochemistry of Ki-67, CD68, FXIIIa
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5
FFPE Immunofluorescence for GPR43 and CD68
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FFPE biopsies were cut 2-3 µm thick and were incubated at 80°C for 30 minutes followed by immersing in Xylol twice for 10 minutes each following descending alcohol line for 5 minutes each. Sections blocked with 20% Bovine Serum Albumin (BSA) for 20 min at room temperature (RT). Single immunofluorescence was performed for GPR43 (Biozol, LSA1578-50, rabbit polyclonal). Double immunofluorescence was performed for GPR43 (Biozol, LSA6599, rabbit polyclonal) and CD68 (Dako, PG-M1, mouse monoclonal). Primary antibodies were diluted in 1% BSA and were applied to biopsy section at the dilution of 1:50 for 1 hour at RT followed by secondary antibodies Alexa Flour (AF) 488 and Alexa Flour 594 (Invitrogen) for 30 minutes (dilution 1:100) in the dark at RT. CD68 was conjugated with AF488 and GPR43 was conjugated with AF594. Sections were counterstained with DAPI and were sealed with mounting media. Biopsy sections were washed three times with PBS after every step. GPR43 positive cells were observed and images were taken at 200X magnification using Zeiss epifluorescence microscope.
6
Immunohistochemical Profiling of Tumor Markers
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Immunohistochemical (IHC) procedures were conducted as described previously [15 (link)]. Antibodies used are PD-L1 (SP142, rabbit monoclonal, Spring Bioscience, Pleasanton, CA, USA), PD-1 (NAT105, mouse monoclonal, GeneTex, Irvine, CA, USA), CD4 (clone SP35, rabbit monoclonal, Spring Bioscience, Pleasanton, CA, USA), CD8 (clone C8/144B, mouse monoclonal, Nichirei Biosciences, Tokyo, Japan), CD68 (PG-M1, mouse monoclonal, DAKO, Tokyo, Japan), and VEGF (A-20, rabbit polyclonal, Santa Cruz, Dallas, TX, USA). For PD-L1 staining, antigen retrieval was done by autoclaving at 121 °C for 10 min in Tris/EDTA buffer (pH 9.0), and 1st antibody incubation (1:100) was conducted at 4 °C overnight. The corresponding normal endometria or stroma provided an internal positive control, and negative controls without addition of primary antibody showed low background staining.
7
Quantifying T Cells and Macrophages in Kidney Capillaries
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The number of CD3+ T cells and CD68+ macrophages in glomerular and peritubular capillaries was assessed by immunoperoxidase staining. Tissue sections were incubated with rabbit polyclonal CD3 (Prediluted, clone 2GV6; Ventana, Tucsan, AZ, USA) or antihuman CD68 (1:50, clone PG-M1; DAKO, Carpenteria, CA, USA) primary antibody. Absolute numbers of intracapillary positive cells in 5 glomerular and peritubular capillaries were counted and mean number of positive cells per glomerular and peritubular capillary was presented.
8
Quantitative Immunohistochemistry of Macrophages, Mast Cells, and Vessels in Adipose Tissue
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Part of each biopsy was fixed in phosphate-buffered formalin, processed (Leica TP 1020, Leica Microsystems), embedded in paraffin, and cut into 5-μm-thick sections. The sections were subjected to high-temperature antigen retrieval for 10 min in EDTA buffer, pH 8, followed by blocking and incubation with antibodies against macrophages (Monoclonal mouse anti-human CD68, clone PG-M1, M0876, Dako, Glostrup, Denmark), mast cells (monoclonal mouse anti-human mast cell tryptase, clone AA1, M7052, Dako), and von Willebrand factor (polyclonal rabbit anti-human von Willebrand Factor, A0082, Dako) to detect vessels. For visualization of immunoreactions, MACH3 mouse AP-polymer (M3M532) was used for macrophages and mast cells and MACH3 Rabbit AP-Polymer (M3R533) for vessels (Biocare Medical, Concord, CA). The chromogen was Warp Red (WR806, Biocare Medical). Slides were counterstained with hematoxylin, mounted, scanned with a Mirax Digital Desk Scanner (Zeiss, Göttingen, Germany), and analyzed with Tissue Studio 3.6.1 (Definiens, Munich, Germany). Macrophage and mast cell densities were expressed as the number of immunoreactive cells per 103 adipocytes. Vessel density was expressed as positive signal area per adipocyte.
9
Monoclonal and Polyclonal Antibody Profiling
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The following monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) were used in this study: rat anti-mouse F4/80 mAb (1:50, clone BM8, T-2028, BMA Biomedicals, Switzerland); rat anti-mouse B7-1/CD80 mAbs (1:100, R&D Systems, clone, 111114; Minneapolis, MN); rabbit anti-mouse CD206 pAbs (1:1000, ab64693, Abcam, Cambridge, UK); mouse anti-human CD68 mAb (1:100, clone PG-M1, M0876, DAKO); rabbit anti-human iNOS pAbs (1:500, GTX124210, GeneTex, Alton Pkwy Irvine, CA); mouse anti-human CD163 mAb (1:200, clone, 10D6; Leica Biosystems, Buffalo Grove, IL); goat anti-mouse IgG (HRP) preabsorbed (1:200, ab97040, abcam, Cambridge, UK); goat anti-rabbit IgG (HRP) preabsorbed (1:200, ab7090, abcam); and goat anti-rat IgG (HRP) preabsorbed (1:200, ab7097, abcam).
10
Multiplex Immunofluorescence Staining Protocol
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To prepare specimens for multiplex IF staining, tissue sections were cut at 4 μm from formalin-fixed paraffin-embedded blocks. All sections on slides were deparaffinized using the Bond ER2 Leica Biosystems AR9640, followed by staining with the Leica Bond RX autostainer. The slides were stained with - CD3 (1:50 SP7 Abcam ab16669), CD16 (1:50 2H7 CellSignaling Technologies 88251S), CD32b (1:2000 NovusBiologicals NBP2–89364), and CD68 (1:400 PGM-1 DAKO). Antibody binding was visualized using secondary anti-mouse/anti-rabbit polymer HRP followed with TSA-Opal reagents (Akoya NEL811001KT). Tissue slides were incubated with DAPI as a counterstain and coverslipped with VectaShield mounting media (Vector Labs). Control tissue samples were stained for each marker as positive controls.
Whole slide digital images were captured with the PhenoImager HT platform and analyzed with QuPath software24 (link). Tissue samples from consecutive slides were stained by conventional H&E and scanned with the Leica SCN400F platform. H&E slides in conjunction with Cytokeratin staining were used to annotate tumor regions within the whole slide image. Cells were segmented, phenotyped, and enumerated using the QuPath object classification algorithm.
Whole slide digital images were captured with the PhenoImager HT platform and analyzed with QuPath software24 (link). Tissue samples from consecutive slides were stained by conventional H&E and scanned with the Leica SCN400F platform. H&E slides in conjunction with Cytokeratin staining were used to annotate tumor regions within the whole slide image. Cells were segmented, phenotyped, and enumerated using the QuPath object classification algorithm.
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