The 4 mm thick FFPE tissues were deparaffinized in xylene before they were rehydrated in gradient concentrations of ethanol. In antigen retrieval step, the samples were incubated in sodium citrate buffer (PH = 6.0) for 4 min. Hydrogen peroxide was used to block the endogenous peroxidase activity for 10 min. The samples were covered with the primary antibodies (ASCL1, Abcam, ab213151; NeuorD1, Abcam, ab60704; YAP1, CST, 14074; POU2F3, Abcam, ab191840; CD44, CST, 3570). After incubation with the secondary antibodies for IHC, the samples were dehydrated, covered with coverslips as previously done.[49 ] For multiplex immunohistochemistry, PANO 4‐plex IHC kit (Panovue, Beijing, China) was used. Panel 1 antibodies (CD3, Abcam, ab17143; CD8, CST, 70306; HAVCR2, CST, 45208), and panel 2 antibodies (CD16, Abcam, ab246222; CD56, CST, 99746S; TGFBR2, Abcam, ab78419) were applied sequentially. And Mantra System (PerkinElmer, Waltham, Massachusetts, US) was utilized to obtain multispectral images.
Pano 4 plex ihc kit
Manufactured by Panovue
Sourced in China
The PANO 4-plex IHC kit is a laboratory equipment product used for simultaneous detection and visualization of up to four target proteins in a single tissue sample using immunohistochemistry (IHC) techniques.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Mantra system, by PerkinElmer (2 mentions)
Ab78419, by Abcam (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Ab213363, by Abcam (1 mentions)
Ab133335, by Abcam (1 mentions)
Cd20 mouse anti human antibody, by Agilent Technologies (1 mentions)
D9542, by Merck Group (1 mentions)
Fap antibody, by Cell Signaling Technology (1 mentions)
Vimentin antibody, by Cell Signaling Technology (1 mentions)
α sma antibody, by Abcam (1 mentions)
11 protocols using pano 4 plex ihc kit
1
Multiplex Immunohistochemistry for Immune and Tumor Markers
2
Multiplex Immunohistochemistry for Tissue Analysis
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Sections prepared from paraffin-embedded tissues were processed for mIHC staining with a PANO 4-plex IHC Kit (Panovue, Beijing, China) following the manufacturer’s instructions. Antigen retrieval was performed by microwaving the sections in 10 mM citrate buffer (pH 6.0). Then, endogenous peroxidase was inactivated by immersion of the sections in 3% hydrogen peroxide for 15 min. For each round of staining, the tissue sections were blocked in 10% normal goat serum for 30 min and then incubated with diluted primary antibodies (anti-Vimentin, 1:1,000; anti-Snai1, 1:500; anti-E-cadherin, 1:2,000; anti-ND1, 1:1,000; anti-COX1, 1:2,000; anti-CYTB, 1:1,000; anti-F4/80, 1:20,000; anti-Ly6G, 1:5,000) for 1 h at room temperature. The secondary antibodies and TSA system were provided in the kit. Finally, the slides were imaged by an SP8 confocal microscope (Leica Microsystems, Buffalo Grove, IL, USA) and analyzed by ImageJ v1.52n (National Institutes of Health, Bethesda, MD, USA).
3
Multiplex Immunohistochemistry of Thymoma
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Thymoma tissue was paraffin-embedded and sectioned. Multiplex staining of thymoma tissue was completed using PANO 4-plex IHC kit (Cat# 10001100100, Panovue) according to the manufacturer’s instruction. The paraffin sections were deparaffinized, dehydrated, antigenically repaired and then closed with 5% BSA, after which the sections were incubated with a primary antibody at 4 °C for one night, then taken out and rewarmed at room temperature, incubated with HRP secondary antibody, washed and then stained by adding the dye of the corresponding panel, and so on, and so forth for several rounds of index staining, and then finally the nuclei of the cells were ransed by using DAPI, and then finally the slices were blocked. CD19 was used to tag the whole B cells. Non-switched B cells were performed by CD19, CD27, and IgD positive expression. Meanwhile, switched memory B cells were CD19 and IgD- positive expressions, which were CD27- negative expressions.
4
Immunohistochemical Analysis of HNSCC
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Sectioning and immunohistochemical (IHC) staining of formalin fixed, paraffin‐embedded (FFPE) HNSCC specimens was performed by the standard protocols. All sections were 5 mm thick. Briefly, sections were deparaffinized through xylenes and graded ethanol, and antigen retrieval was performed in Tris/EDTA buffer at pH 9.0. For multiple immunofluorescence staining, PANO 4‐plex IHC kit (PANOVUE) was used according to the manufacturer's instructions. The samples were then observed under a Leica TSC‐SP2 Microsystems. Primary antibodies against CD14 (ab133335), CD68 (ab213363) were purchased from Abcam.
5
Multiplex Immunofluorescence Staining Protocol
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Multiplex immunofluorescence staining was performed using PANO 4-plex IHC kit (cat 10001100100, Panovue). We performed the fluorescent dyes by using the CD20 mouse anti-human antibody (Dako, M0755), CD86 rabbit anti-human antibody (CST, 91882S), and FCRL4 rabbit anti-human antibody (Abcam, ab239076). Different above primary antibodies were applied, followed by horseradish peroxidase-conjugated secondary antibody incubation and tyramide signal amplification. The slides were microwave heat-treated after each TSA operation. Nuclei were stained with DAPI (SIGMA-ALDRICH, D9542) after all the human antigens had been labelled. To obtain multispectral images, the stained slides were scanned using the Mantra System (PerkinElmer, Waltham, Massachusetts, USA), which captures the fluorescent spectra at 20-nm wavelength intervals from 420 to 720 nm with identical exposure time; the scans were combined to build a single stack image.
6
Multiplex Immunofluorescence Staining of FFPE Thyroid Tissues
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FFPE GD thyroid tissues (n=4) were cut into 4-μm sections for immunofluorescence staining. Multiplex immunofluorescence staining was performed by using a PANO 4-plex IHC kit (Panovue, China) according to the manufacturer’s instructions. Briefly, sections were deparaffinized and then subjected to antigen repair. The antigen repair conditions were the same as immunohistochemical staining for human CD11c and CD19. Then, primary antibodies were sequentially applied after blocking, followed by HRP-conjugated antibody incubation. The details and usage of primary antibodies and the fluorescent dye in kit were provided in Supplementary Table S2
7
Multiplex IHC Analysis of CD8+ and CD86+ Cells in TME
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To evaluate the density of the CD8+ and CD86+ cell composition of the TME and their relative spatial positioning in tumors, the PANO 4-plex IHC kit (catalog No. 10079100100, Panovue) was utilized for multiplex staining of tissue that was formalin-fixed and then embedded in paraffin. Following sequential application of CD8 (catalog No. 98941, CST) and CD86 (catalog No. 19589, CST) antibodies, horseradish peroxidase-conjugated secondary antibodies were incubated and tyramide signal amplification was performed. Following each tyramide signal amplification operation, the slides were microwave-heated. The DAPI stain was applied after all antigens were labeled. Multispectral images from the stained slides were acquired at 10× and 40× magnification using Zeiss Axio Imager Z2 multispectral microscope within the same exposure time. Each slide was scanned in five random areas without necrosis or damage.
8
Immunohistochemical Analysis of FGF2 Expression
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Paraffin-embedded tumor samples were provided by the Sun Yat-sen University Cancer Center under a protocol approved by the institutional review board. The IHC study was performed using a standard streptavidin-biotin-peroxidase complex method as described in a previous publication (21) . An immunoreactivity scoring system was applied to analyze FGF2 IHC staining. The intensity of FGF2-positive staining was scored as: 0, negative; 1, weak; 2, moderate; or 3, strong. Opal multicolor immunofluorescence (IF) staining was performed using a PANO 4-plex IHC Kit (Panovue).
9
Multiplex Immunofluorescence Staining of CD90 and RANKL
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Multiplex immunofluorescence staining was performed using PANO 4-plex IHC kit (cat 10,001,100,100, Panovue). We performed the fluorescent dyes by using the Mouse anti Human CD90 antibody, clone F15-42–1 (Dako, MA5-16,671), and RANKL rabbit anti-human antibody (PA5-110,268). After applying different primary antibodies, horseradish peroxidase-conjugated secondary antibody incubation and tyramide signal amplification were conducted. Following this, the slides were microwaved heat-treated. After labeling all human antigens, DAPI (SIGMA-ALDRICH, D9542) was used to stain the nuclei. Fluorescent images were captured by Confocal microscopes Leica SPE (Leica).
10
Multicolor Immunofluorescence Staining of Cells and Tissues
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Cells grown on slides were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, and then permeabilized with 0.1% Triton X‐100 and blocked with 4% BSA‐PBS. Next, slides were incubated with α‐SMA antibody (Abcam; 1:100), FAP antibody (Cell Signaling Technology; 1:200), vimentin antibody (Cell Signaling Technology; 1:200), and SLPI antibody (Novus; 1:200) overnight at 4°C. After that, the cells were incubated with fluorescence‐conjugated secondary antibody and subjected to DAPI staining. Images were photographed under a confocal fluorescence microscope. For tissues, multicolor immunofluorescence staining of SLPI, Ki67, FAP, and DAPI was performed using a PANO 4‐plex IHC kit (Panovue, China) according to the manufacturer's instructions. In brief, paraffin‐embedded tissue sections were baked in a dry oven for 60 min at 60°C, followed by deparaffinization and rehydration. After antigen retrieval and blocking, sections were washed three times and incubated with primary antibody, followed by the incubation with biotinylated secondary antibody and tyrosine signal amplification diluent. During each round of staining, the slides were subjected to high‐pressure treatment. After all antigens were labeled, the nucleus was subjected to DAPI staining. Images were obtained using a Vectra Polaris slide scanner (PerkinElmer).
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