Tissue samples were fixed in 10% Formalin and embedded in paraffin wax (Leica RM2255 microtome). Sections were cut in Palm slides and underwent Haematoxylin&Eosin (H&E) staining and automated Immunohistochemistry staining (Leica ST5010). Histology slides were imaged on a Pannoramic 250 Slide Scanner (3DHISTECH) and images exported to ImageJ. Images were converted to RGB stacks and a threshold was set across all images to identify positive staining. H&E and PAX8 staining was used to identify HGSC tissue. Regions of interest (ROIs) were then manually drawn around discrete areas of HGSC tissue. The data were reported as the total area percentage of the ROI staining positive. PAX8: Abcam ab13611, 1:100; F4/80: Biorad MCA497GA, 1:1000; Adenovirus: Abcam ab8251, 1:1000; DX5: Abcam ab133557, 1:250.
Pannoramic 250 slide scanner
Manufactured by 3DHISTECH
Sourced in Hungary
The Pannoramic 250 is a digital slide scanner manufactured by 3DHISTECH. It is designed to digitize and create high-resolution digital images of glass microscope slides. The scanner uses advanced optical and imaging technologies to capture detailed, high-quality digital representations of the slides.
Automatically generated - may contain errors
Lab products found in correlation
Pannoramic viewer, by 3DHISTECH (2 mentions)
Alexa fluor 488 goat anti human igg h l, by Thermo Fisher Scientific (2 mentions)
Liquid dab substrate chromogen system, by Agilent Technologies (2 mentions)
Ab8251, by Abcam (1 mentions)
Ab133557, by Abcam (1 mentions)
Rm2255 microtome, by Leica (1 mentions)
St5010, by Leica (1 mentions)
Mca497ga, by Abcam (1 mentions)
Trisodium citrate dehydrate, by Merck Group (1 mentions)
Citric acid monohydrate, by Merck Group (1 mentions)
Click it tunel alexa fluor imaging assay, by Thermo Fisher Scientific (1 mentions)
Vectastain abc and dab peroxidase substrate kits, by Vector Laboratories (1 mentions)
Axioskop 2, by Zeiss (1 mentions)
Axiocam hrc camera, by Zeiss (1 mentions)
Clone 5a1e, by Cell Signaling Technology (1 mentions)
Ncl l ki67 mm1, by Cell Signaling Technology (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Axio imager m1 microscope, by Zeiss (1 mentions)
Tissue freezing medium, by Leica (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
29 protocols using pannoramic 250 slide scanner
1
Histological Analysis of HGSC Tissue
2
Histological Analysis of Colonic Inflammation
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Formalin fixed tissues were embedded in paraffin and cut in longitudinal 4 µm thick sections prior to hematoxylin & eosin (HE) staining. All samples were scanned using a Pannoramic 250 Slide scanner (3DHistech, Budapest, Hungary) with 20× objective and analyzed with Pannoramic viewer (3D Histech, software version 1.15.4). Crypt length was quantitated as an average from 3–5 crypts; severity of colonic inflammation was scored with a score of 0 = healthy, 1 = some inflammatory cells seen in mucous, 2 = moderate infiltration, and 3 = a severe and acute inflammation; edema scoring was based on location and severity of edema and ranged from 0–3; erosion depth was scored as 0 = no erosion, 1 = erosion of epithelium, 2 = erosion of mucous, 3 = erosion going through muscular lamina; hyperproliferation was scored from 0–3 based on both the amount of elongated crypts (where 0 was normal crypt length and 3 longest crypt lengths or missing crypts) and the loss of goblet cells together with the amount of mitotic cells (where 0 = no change, 1 = weak, 2 = moderate, and 3 = severe effect/lot) [35 (link)]. Two people did the scoring independently and blind for the sample group. Presented scores are averages of these two analyses.
3
Immunohistochemical Detection of Androgen Receptor
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Formalin-fixed and paraffin-embedded tumor samples were cut to sections prior to deparafinization and rehydration. The sections were exposed to the antigen retrieval in a steamer in 10 mM sodium citrate buffer (Citric acid monohydrate Sigma-Aldrich, St Louis, USA) and Tri-sodium citrate dehydrate (Merck, Darmstadt, Germany) for 30 min. The sections were then incubated in a humidified chamber overnight with the primary antibody against the N-terminus of the AR (N-20: sc-816, dilution 1:250, Santa Cruz Biotechnology, Dallas, Texas, USA) at 4 °C. Endogenous peroxidase activity was blocked by applying 1% H2O2 for 20 min at room temperature and the section were then incubated for 30 min with anti-rabbit antibody conjugated with polymer-HRP (Dako, Glostrup, Denmark), washed and visualized with Envision+ System-HRP DAB staining (Dako). The sections were counterstained with hematoxylin, mounted and digitized using a Pannoramic 250 slide scanner (3DHISTECH, Budapest, Hungary).
4
Immunofluorescence Analysis of Pancreatic Tissue
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Pancreata were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 3.5 μm sections. Antigen retrieval was performed with a 10 mM sodium citrate buffer (pH 6.0). Sections were permeabilized and blocked in phosphate buffered saline (PBS) containing 0.1% Triton-X-100, 1% bovine serum albumin (BSA), and 5% donkey or goat serum. Primary antibody binding was performed overnight at 4 °C, whereas secondary antibody incubation was performed at room temperature for 1 h. Slides were scanned using a 20× objective on the Pannoramic 250 Slide scanner (3D Histech). TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining was performed using the Click-iT TUNEL Alexa Fluor Imaging Assay (Invitrogen) with the following modification: after deparaffinization, slides were incubated with Proteinase K for 15 min, rinsed, and immersed in 4% paraformaldehyde for 5 min before continuing with the protocol. Antibodies used for immunofluorescence staining are listed in Supplemental Table 2 .
5
Immunohistochemical Analysis of Wound Sections
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PFA-fixed paraffin sections of human and mouse wounds were dewaxed and rehydrated using a xylene/ethanol gradient followed by antigen retrieval using citrate buffer (10 mM citric acid, pH 6.0) at 95°C for 1 hour. Unspecific binding sites were blocked with 12% BSA in PBS for 1 hour at room temperature. Incubation with primary antibodies (table S4) at 4°C O/N was followed by incubation with biotin-conjugated secondary antibodies (table S5). After each antibody incubation step, extensive washing steps in 0.1% Tween in PBS were performed (3 × 10 min). The VECTASTAIN ABC and DAB peroxidase substrate kits (#PK-6100 and #SK-4100; Vector Laboratories, Burlingame, CA) were used for signal visualization according to the manufacturer’s instructions. Sections were counterstained with hematoxylin and eosin (65 (link)) and mounted with Eukitt. Immunohistochemistry sections were imaged using a 3DHistech Pannoramic 250 slide scanner (3DHistech, Budapest, Hungary).
6
Fluorescence and Histochemical Imaging
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Fluorescence stainings were imaged using an Axioskop 2 fluorescence microscope (Carl Zeiss, Inc., Oberkochen, Germany), and the corresponding software (Carl Zeiss, Inc.). Image acquisition was performed with an Axiocam HRc camera (Carl Zeiss, Inc.) connected to the microscope. Histochemical stainings were imaged with a Pannoramic 250 slide scanner (3DHISTECH, Budapest, Hungary). Quantifications were performed using the Fiji software [19 (link)].
7
Collagen Quantification in Small Intestine
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Small intestinal tissue was fixed in 4% paraformaldehyde and paraffin-embedded overnight. The small intestine sections were cut into two μm thick and soaked overnight in ZGSJ (Masson A). The small intestine sections were stained with Weigert’s hematoxylin (Masson A and Masson B in equal amounts) for 1 minute, differentiated by 1% acid ethanol, Then it was stained in scarlet magenta solution (Masson D) for 6 min, differentiated in phospho-molybdenum-phosphotungstate solution (Masson E) for 1 min, and transferred directly (without rinsing) to aniline blue solution (Masson F) for 2-30s. Then, rinse briefly in distilled water for 2-5 minutes. Finally, dehydration through anhydrous ethanol, xylene transparent, and neutral sealant. The sections were scanned by Pannoramic 250 slide scanner (3D HISTECH). Micrographs were analyzed by the blind method. Collagen volume fraction (CVF) was observed by image analysis system software (HALO, Indica Labs, American). Technical support was provided by Servicebio, Inc. (Wuhan, China).
8
Quantifying Cell Proliferation and Apoptosis in Tumor Explants
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Explant cultures were embedded in paraffin and cut into 5 μM sections, after which hematoxylin and eosin (HE) staining was performed using standard methods. For immunohistochemistry, the sections were deparaffinized, blocked with 3% hydrogen peroxide and 10 mM citrate buffer, incubated with antibodies directed against Ki67 (Novocastra, NCL-L-Ki67-MM1) or cleaved caspase 3 (Cell Signaling Technologies, clone 5A1E, #9664) at 4 °C o/n, and with a biotinylated secondary antibody for 1 h at room temperature. Next, a chromogenic HRP-mediated diaminobenzidine staining method was used (Vectastain Elite ABC Kit, Vector Laboratories, USA), after which the samples were counterstained with Mayer’s hematoxylin. The stained sections were scanned using a digital slide scanner (Pannoramic 250 Slide Scanner, 3DHistech). Five representative images from cancerous areas were taken at 40x magnification. The number of stained cells/nuclei was quantified using thresholding within the Image J program, and the total number of tumor cells was calculated manually.
9
Histological Wound Analysis Protocol
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Wounds were excised and fixed overnight with either 4% paraformaldehyde (PFA) or 95% ethanol/1% acetic acid, followed by tissue processing and paraffin embedding, or they were directly frozen in tissue freezing medium (Leica Microsystems, Heerbrugg, Switzerland). Sections of 7 μm thickness from the middle of the wounds were stained with hematoxylin and eosin (H&E) or with Herovici stain [17 (link)] or used for further immunohistochemistry/immunofluorescence analysis. Stained sections were photographed using a Zeiss AxioImager.M1 microscope equipped with Zen Pro software (Zeiss, blue edition, 3.2) (Carl Zeiss AG, Oberkochen, Germany) to control the Axiocam MRm camera or using the Pannoramic 250 Slide Scanner (3D Histech, Budapest, Hungary). Analysis of the various wound parameters [15 (link)] and staining quantifications were performed using ImagePro® Plus software (Media Cybernetics Inc., Rockville, MD, USA).
10
Histological Analysis of Mouse Tissues
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Pieces of PC from 2–5-mo-old male and female mice were fixed with 4% neutral formalin and embedded in paraffin. The samples were sectioned into 4-µm-thick longitudinal sections, mounted on microscope slides and stained using hematoxylin & eosin (H&E). The stained samples were scanned using a Pannoramic 250 slide scanner (3DHISTECH, Budapest, Hungary) with a 20× objective. CaseViewer software (3DHISTECH, software version 2.4) was used for creating images for analysis.
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