IHC was used to detect PKM2 staining as previously described [13 (link)]. In brief, the sections were stained with an anti-PKM2 antibody (Cell Signalling Technology, 1:1000) and incubated overnight at 4°C. The sections were then processed using a MaxVision™ HRP-Polymer Anti-Rabbit IHC Kit (Maixin, Fuzhou, China), developed with a DAB Horseradish Peroxidase Colour Development Kit (Maixin, Fuzhou, China) and counterstained with haematoxylin. The degree of immunostaining was scored according to both the proportion of positively stained tumour cells (0∼3) and the staining intensity (0∼3), as previously described [29 (link)]. The staining index was calculated by multiplying the staining intensity score by the proportion of positive tumour cells, and the results were 0, 1, 2, 3, 4, 6, and 9. An optimal cut-off value (median) was identified as follows: a staining index score of >4 was used to define high PKM2 expression, and a score ≤4 was defined as low PKM2 expression.
Maxvision hrp polymer anti rabbit ihc kit
Manufactured by Maixin Group
Sourced in China
MaxVision™ HRP-Polymer anti-Rabbit IHC Kit is a laboratory equipment product designed for immunohistochemistry (IHC) applications. It utilizes a horseradish peroxidase (HRP)-polymer detection system to facilitate the visualization of rabbit primary antibodies in tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Dab horseradish peroxidase colour development kit, by Maixin Group (2 mentions)
Anti zfp36l1, by Abcam (2 mentions)
Dab horseradish peroxidase color development kit, by Maixin Group (2 mentions)
Anti pkm2 antibody, by Cell Signaling Technology (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Leica dm4000b, by Leica Microsystems (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Vimentin, by Bioss Antibodies (1 mentions)
Rabbit anti vimentin, by Bioss Antibodies (1 mentions)
Dab kit, by Maixin Group (1 mentions)
8 protocols using maxvision hrp polymer anti rabbit ihc kit
1
PKM2 Immunohistochemistry Staining Protocol
2
Immunohistochemical Analysis of Cleaved Caspase-3
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Immunohistochemical staining for cleaved caspase-3 was performed on 5-µm thick sections embedded in paraffin after formalin fixation. The sections were de-paraffinized in xylene and rehydrated in graded ethanol solutions (reducing concentration from 95 to 70%). The sections were incubated in H2O2 solution (3% H2O2 in PBS buffer) for 30 min to block endogenous peroxidase activity. Antigen retrieval was performed in retrieval buffer (pH 9.0, 20 mM Tris, 0.05 mM EDTA, 0.05% Tween-20 buffer) in a 99°C bath for 20 min. The sections were subsequently incubated at 4°C overnight with anti-cleaved caspase-3. After rinsing with PBS buffer, the secondary antibody (MaxVision™ HRP-Polymer Anti-Rabbit IHC kit; Maixin Bio, Beijing, China) was applied for 15 min at room temperature (RT). The DAB (Maixin Bio) solution was used as the chromogen. Finally, the sections were counterstained with hematoxylin (Sigma-Aldrich) to identify the nuclei. The images were captured and analyzed using a microscope (Leica DM4000 B; Leica Microsystems GmbH, Wetzlar, Germany) with Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).
3
Immunohistochemical Quantification of HK2 in Tumors
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Immunohistochemistry (IHC) was used to detect HK2, as previously described [12 (link)]. Briefly, paraffin sections were deparaffinized using xylene and rehydrated using a series of alcohol solutions. Antigen retrieval was performed by treating the sections with boiling citrate buffer (pH 6.0) for 4 min in a pressure cooker. Endogenous peroxidase activity was blocked by treatment with 3% H2O2 for 15 min. The sections were then incubated with an anti-HK2 antibody (Atlas, 1:250) overnight at 4°C. The sections were incubated with the secondary antibody using a MaxVision™ HRP-Polymer anti-Rabbit IHC Kit (Maixin, Fuzhou, China); colour was developed using a DAB Horseradish Peroxidase Colour Development Kit (Maixin, Fuzhou, China), and the sections were counterstained with haematoxylin. The degree of immunostaining was scored according to both the proportion of positively stained tumour cells and the staining intensity, as described previously [30 (link)]. We evaluated HK2 expression by determining the staining index (staining intensity score × proportion of positive tumour cells), which resulted in scores of 0, 1, 2, 3, 4, 6 and 9. An optimal cut-off value (median) was identified, with a staining index score of >4 defining a high level of HK2 expression and a staining index score of ≤4 defining a low level of HK2 expression in the tumours.
4
Immunostaining Protocol for G9A Protein
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Immunostaining was performed as described previously (25 (link)). Briefly, deparaffinized sections were treated with 3% H2O2 and subjected to antigen retrieval by citric acid (pH 6.0). Subsequent to overnight incubation with primary antibody of G9A (1:100; cat. no. ab133482; Abcam, Cambridge, UK) at 4°C, the sections were incubated for 15 min at room temperature with horseradish peroxidase-labeled polymer conjugated with secondary antibody (MaxVision™ HRP-Polymer anti-Rabbit IHC Kit; Maixin-Bio, Fuzhou, China) and incubated for 1 min with diaminobenzidine. The sections were then lightly counterstained with hematoxylin. The sections without primary antibody served as negative controls. The positive brown staining was visualized and then photographed using a light microscope at ×200 or ×400 magnification (BX51, Olympus Corporation, Tokyo, Japan).
5
Evaluating Tumor Cell Proliferation
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The tumor tissues were evaluated for cell proliferation using Rabbit anti-human Ki-67 monoclonal antibody (Maixin Biotech, Fuzhou, China). Rabbit Anti-P-CK/Cytokeratin AE1 + AE3 (Bioss, Beijing, China) (CK) and Rabbit Anti-Vimentin (Bioss, Beijing, China) were used to determine the origin of cells in the tumor tissue, CK-positive epithelial cells, or Vimentin-positive stromal cells. Formalin-fixed, paraffin-embedded tissue blocks were cut into 5-μm sections and mounted on positively charged slides. Tissue sections were deparaffinized with xylene and rehydrated with graded alcohol solutions; they were then incubated in citrate buffer (pH 6.0) at 210°C for 10 min and at 160°C for 10 min in a pressure cooker. After incubation in 3.0% hydrogen peroxide for 10 min and PBS wash, the tissue sections were immersed in working solution of anti-Ki-67, anti-cytokeratin, or anti-Vimentin for 1 h at 37°C. Tissue sections were exposed with a second antibody (MaxVision™ HRP-Polymer anti-rabbit IHC kit, Maixin Bio, Fuzhou, China) for 15 min at room temperature. Finally, the sections were incubated in DAB chromogen and then counterstained with hematoxylin. Positive and negative controls were used throughout all immunostaining protocols. The ratio of Ki-67-positive cells was defined as the percentage of positive cancer cells by counting 2000 cancer cells at ×200 microscopically.
6
Immunohistochemical Analysis of ZFP36L1
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This procedure was performed as previously described [16 (link), 17 (link)]. In short, paraffin sections were deparaffinized using xylene and rehydrated using a series of alcohol solutions. Endogenous peroxidase activity was blocked by treatment with 3% H2O2 for 15 min. Then the sections were incubated with an anti-ZFP36L1 (Abcam, 1:100) antibody overnight at 4 °C. After washing with PBS three times, the tissue slides were treated with secondary antibody using a MaxVision™ HRP-Polymer anti-Rabbit IHC Kit (Maixin, China). The color was developed using a DAB Horseradish Peroxidase Color Development Kit (Maixin, China), and the sections were counterstained with haematoxylin. The degree of immunostaining was scored according to both the proportion of positively stained tumor cells and the staining intensity. The score for each tissue was calculated by multiplying the staining index (0, 1, 2 and 3) by the percentage category value (0, 1, 2, 3 and 4), and the average of the scores from the two pathologists was used as the final score.
7
Immunostaining of RCC Tissue Sections
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Immunostaining of the paraffin-embedded RCC tissue sections was performed by a similar method to that used in our previous work [36 ]. The sections were deparaffinized and rehydrated, then boiled for the antigen retrieval. The endogenous hydrogen peroxidase was blocked. After being incubated with 10% BSA, the sections were incubated with anti-C12orf59 antibody (HPA036147, Sigma, USA) used at a 1:300 dilution at 4°C overnight. After being washed in PBS, the sections were treated with a MaxVision HRP-Polymer anti-rabbit IHC Kit (Maixin Bio, Fujian, China) and stained with a DAB kit (Maixin Bio, Fujian, China). The expression of C12orf59 was assessed blindly by two independent investigators. The staining of C12orf59 was scored as the product of the staining intensity (on a scale of 0–3: negative = 0, weak = 1, moderate = 2, strong = 3) and the percentage of cells stained (on a scale of 1–5: 1 = 0%–20%; 2 = 21–40%; 3 = 41–60%; 4 = 61–80%; 5 = 81%–100%), resulting in scores ranging from 0 to 15. We defined two subgroups as follows: the low expression group (scores: 0–5) and the high expression group (scores: 6–15) [37 (link)].
8
Immunohistochemical Analysis of ZFP36L1 Expression
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This procedure was performed as previously described [16, 17] . In short, para n sections were depara nized using xylene and rehydrated using a series of alcohol solutions. Endogenous peroxidase activity was blocked by treatment with 3% H 2 O 2 for 15 min. Then the sections were incubated with an anti-ZFP36L1 (Abcam, 1:100) antibody overnight at 4°C. After washing with PBS three times, the tissue slides were treated with secondary antibody using a MaxVision™ HRP-Polymer anti-Rabbit IHC Kit (Maixin, China). The color was developed using a DAB Horseradish Peroxidase Color Development Kit (Maixin, China), and the sections were counterstained with haematoxylin. The degree of immunostaining was scored according to both the proportion of positively stained tumor cells and the staining intensity.
The score for each tissue was calculated by multiplying the staining index (0, 1, 2 and 3) by the percentage category value (0, 1, 2, 3 and 4), and the average of the scores from the two pathologists was used as the nal score.
The score for each tissue was calculated by multiplying the staining index (0, 1, 2 and 3) by the percentage category value (0, 1, 2, 3 and 4), and the average of the scores from the two pathologists was used as the nal score.
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