Tissue sections <1 cm3 were obtained from fresh cervical CIN and cervical cancer tissue samples, fixed in 10% neutral formalin at room temperature for 24 h, then dehydrated, embedded in paraffin, deparaffinized and rehydrated in graded ethanol (100, 95, 85 and 75%). Antigen retrieval was performed by heating the slides (10 min in a microwave oven, 122 mm) in citrate buffer at pH 6.0. Endogenous peroxidase activity was blocked with 0.3% H2O2 for 10 min at room temperature. Sections were incubated with primary antibodies at 1:200 dilutions (anti-XIAP; A-7: sc-55550) and anti-Smac (V-17: sc-12683); Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C, washed with PBS and re-incubated with a secondary antibody horseradish peroxidase (32230; dilution, 1:500; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 30 min at 37°C. Diaminobenzidine staining was performed under close monitoring for 5 min at room temperature. Slides were finally counterstained with hematoxylin at room temperature for 2 min and dehydrated in graded ethanol (75, 85, 95 and 100%). Finally, the slides were imaged using an AperioScanScope GL (Aperio Technologies, Vista, CA, USA) at ×400 magnification.
Aperio scanscope gl
Manufactured by Leica Biosystems
Sourced in United States
The Aperio ScanScope GL is a high-performance digital slide scanner designed for whole slide imaging. It captures high-resolution digital images of microscope slides, enabling digital pathology workflows.
Automatically generated - may contain errors
Lab products found in correlation
Aperio imagescope software, by Leica Biosystems (3 mentions)
Hematoxylin counterstaining, by Merck Group (2 mentions)
Goat serum, by Vector Laboratories (2 mentions)
Ab194201, by Abcam (1 mentions)
Ab51129, by Abcam (1 mentions)
Diaminobenzidine colorimetric reagent solution, by Agilent Technologies (1 mentions)
Horseradish peroxidase conjugated secondary anti mouse or anti rabbit antibodies, by Santa Cruz Biotechnology (1 mentions)
Image pro analyzer software, by Media Cybernetics (1 mentions)
Avidin biotin blocking kit, by Thermo Fisher Scientific (1 mentions)
Vectastain elite abc reagent, by Vector Laboratories (1 mentions)
Harris modified hematoxylin, by Thermo Fisher Scientific (1 mentions)
Immpact dab chromogen, by Vector Laboratories (1 mentions)
Aperio scanscope at turbo, by Leica Biosystems (1 mentions)
12 protocols using aperio scanscope gl
1
Immunohistochemical Analysis of XIAP and Smac in Cervical Cancer
2
Immunohistochemical Analysis of CSE and CBS
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For the immunohistochemical assay, paraffin sections of colonic muscle tissues from rats in the different groups were placed in 60°C for 2 h, followed by incubation with dimethylbenzene for dewaxing. The sections were then hydrated with different concentrations of alcohol (95% for 2 min, 85% for 2 min and 75% for 5 min) and washed with ddH2O for 2 min. Sections were then fixed using 3% H2O2 for 15 min and washed with PBS three times. They were then incubated with primary antibodies against CSE (Abcam, diluted 1:200) and CBS (Abcam, diluted 1:500) at 37°C for 30 min and incubated at 4°C overnight. After three cycles of 0.01 M PBS washes, 5 min for each cycle, the secondary antibody (1:200) was added to the sections and placed at 37°C for 30 min followed by another five cycles of PBS washes. HRP-labelled avidin was then added and incubated with the sections at 37°C for 30 min before addition of DAB. The reaction was kept for 3–10 min and then stopped by ddH2O. Slides were re-stained using haematoxylin and dehydrated. Finally, the slides were scanned using an Aperio ScanScope GL (Aperio Technologies, Vista, CA, U.S.A.) at 200× magnification.
3
Immunohistochemical Assay for Paraffin Sections
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For immunohistochemical assay, paraffin sections from patients were placed at 60°C for 2 h before incubation with dimethylbenzene for dewaxing. The sections then were hydrated with different concentrations of alcohol (95% for 2 min, 85% for 2 min and 75% for 5 min.) and washed with ddH 2 O for 2 min. Afterwards, sections were fixed using 3% H 2 O 2 for 15 min and washed with PBS for three times. They were then incubated with primary antibody (1:50) at 37°C for 30 min before incubation at 4°C overnight. After three cycles of 0.01 M PBS wash, 5 min for each cycle, secondary antibody (1:200) was added to the sections and placed at 37°C for 30 min before another five cycles of PBS wash. HRP-labelled avidin was then added and incubated with the sections at 37°C for 30 min before addition of DAB. The reaction was carried out for 3-10 min and then stopped by adding ddH 2 O. Slides were restained using haematoxylin and dehydrated. Scores of the immunohistochemical assay were determined by scanning the slides using an Aperio ScanScope GL (Aperio Technologies, Vista, CA, USA) at 200 × magnification.
4
Immunohistochemical Localization of IGF2BP1
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The HCC sections were incubated at 60°C for 2 h before incubation with dimethylbenzene for dewaxing. The sections were then hydrated with different concentrations of alcohol (95% for 2 min, 85% for 2 min, and 75% for 5 min) and washed with ddH2O for 2 min. Subsequently, the sections were fixed using 3% H2O2 for 15 min and washed with phosphate-buffered saline (PBS) three times, followed by incubation with a primary antibody IGF2BP1 (at 37°C for 30 min and then at 4°C overnight. After three washes with 0.01 M PBS (5 min each), a secondary antibody (HRP) was added to the sections for 30-min incubation at 37°C, followed by five cycles of PBS wash. Thereafter, the sections were incubated with HRP-labeled avidin for 30 min at 37°C, and reacted with DAB for 3–10 min before the reaction was stopped by ddH2O. The sections were re-stained with haematoxylin and dehydrated. The results were determined by scanning the sections using an Aperio ScanScope GL (Aperio Technologies, Vista, CA, USA) at ×40 magnification.
5
Immunohistochemical Evaluation of XIAP/Smac
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XIAP/Smac immunoreactivity was evaluated by two pathologists come from the First Affiliated Hospital of Wenzhou Medical University blind to the procedure. To further validate the staining of XIAP/Smac in tumor cells, the expression intensity was graded according to the intensity of positive control and the percentage of positive tumor cells. A total of 6 fields of view were randomly selected and analyzed. The slides were first assessed for expression intensity (0, negative; 1, less intense compared with positive control; 2, equal intensity to control; 3, more intense compared with control). Subsequently, the slides were assessed for the rate of positive cells (0, <5%; 1, 5–25%; 2, 26–50%; 3, >50%; magnification, ×400) using an AperioScanScope GL (Aperio Technologies). The multiplication product of two points was used as the final assessment [0, negative (−); 1–4, weakly positive (+); 5–8, moderate positive (++); 9–12, strong positive (+++)].
6
Immunohistochemical Analysis of CBX6, PCNA, and Ki67
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Paraffin-embedded tissue sections and TMAs underwent IHC analyses. Briefly, the slides were probed with primary antibodies specific for the following proteins CBX6, PCNA, and Ki67, and then the slides were treated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Finally, the slides were stained with diaminobenzidine (DAB) colorimetric reagent solution from Dako (Carpinteria, CA, USA) before undergoing hematoxylin counterstaining (Sigma Chemical Co). TMA analysis was performed by scanning the slides with an AperioScanScope GL, and AperioImageScope software (Aperio Technologies, Vista, CA, USA) was used to assess the scanned images by determining the percentages of positively stained cells and staining intensities. CBX6expression in all the clinical samples was quantified, and the tumor CBX1-8 expression level/peri-tumor CBX1-8 expression level ratio was calculated.
7
Renal Tissue Immunohistochemistry Protocol
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The expression levels of HIF-1α, VEGF, CD31, and αSMA in the renal tissues were detected by immunohistochemistry (IHC). The tissue sections were hydrated in serial concentrations of alcohol as follows: 70% for 2 h, 80% overnight, 90% for 2h, 100% for 1, and 100% for 1 h. Afterwards, the sections were placed in dimethylbenzene and paraffin for 30 min before transferring into a 60°C incubator to form paraffin sections. The sections were then fixed in methanol solution with 3% H 2 O 2 for 15 min at room temperature, and washed with PBS for three times with 5 min each time. The sections were incubated with primary antibodies (1:100) against different proteins of interest at 4°C overnight. After washing with 0.01 M PBS for 4× 5 min, the sections were incubated with HRP labeled secondary antibodies (1:200) at 37°C for 30 min, followed by washing with PBS. DAB was added to the sections for 3-10 min reaction, which was stopped by ddH 2 O. The sections were re-stained with haematoxylin and dehydrated. IHC results were assessed by scanning the sections using Aperio ScanScope GL (Aperio Technologies, Vista, CA, USA) at 400× magnification.
8
Immunostaining and Quantification of 14-3-3ζ and p-Akt in HCC
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Matched pairs of primary HCCs and adjacent liver tissues were used for the construction of TMAs (in collaboration with the Shanghai Biochip Company, Shanghai, China) as previously described [30] . Immunostaining was performed on TMA slides following the routine protocol. Rabbit anti-human 14-3-3ζ polyclonal antibody (ab51129, Abcam) and rabbit anti-human AKT1 (phospho-S473) monoclonal antibody (ab194201, Abcam) were used to detect 14-3-3ζ and p-Akt. The slides were scanned with an Aperio ScanScope GL (Aperio Technologies, Vista, CA), and the protein levels were scored by the Aperio ImageScope software (Aperio Technologies) based on the percentage of positively stained cells and the staining intensity. The scores equal to or higher than the median of all values were defined as high expression (14-3-3ζ high or p-Akt high ) while the scores lower than the median were defined as low expression (14-3-3ζ low or p-Akt low ).
9
Immunohistochemical Analysis of Cancer Biomarkers
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Formalin fixed and paraffin-embedded sections (4 μm) were subjected to immunohistochemical staining. The slides were incubated overnight at 4 °C with primary antibodies, including rabbit anti-AHRR (1:100, ab108518, Abcam), rabbit anti-PTPRD(1:100, LS-B9625, LifeSpan Biosciences), rabbit anti-NRG3(1:200, ab83704, Abcam) and mouse anti-UNC5D (1:100, ab58141, Abcam). Anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were applied. Finally, diaminobenzidine colorimetric reagent solution from Dako (Carpinteria, CA) was used and followed by hematoxylin counterstaining (Sigma Chemical Co). Tissue slides were scanned with an Aperio ScanScope GL, and the Aperio ImageScope software (Aperio Technologies, Vista,CA) was used to assess the scanned images based on the percentage of positively stained cells and staining intensity. Expression levels of these proteins in all clinical samples were quantified.
10
Quantifying Iba1 and GFAP Immunoreactivity
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