H&E-stained slides of the most invasive part of primary tumor were used to determine the DR types. All these slides were scanned using digital WSI scanning systems (Aperio AT2, Leica; Aperio GT 450 Leica; MoticEasyScan Pro, Motic; KF-PRO-020, KFBIO; SQS-600P, TEKSQRAY; NanoZoomer S60) at 40 × magnification (resolution: 0.21–0.26 μm /pixel). Image annotation was carried out using the ImageScope software (ImageScope v12.4.3, Leica). Subsequently, 620 slides were selected for IHC. A series of steps were performed, deparaffinage, antigen retrieval solution (using 10 × concentrate solution, Novocastra, Leica) and primary [human anti-CD3 (Gene Tech, catalog no. GT200229) rabbit mAbs] and secondary (rabbit-anti-mouse IgG, Bond Refine Detection Kit, Leica) antibodies, according to the manufacturer's recommendations in a Ventana BenchMark automated staining system. Finally, the sections were incubated with 3,3-Diaminobenzidine, counterstained with hematoxylin, and mounted using special glue. To guarantee quality assurance, an internal positive control was utilized. The IHC-stained tissue sections were then captured utilizing a digital whole-slide scanning system (Aperio AT2, Leica) at 40 × magnification.
Aperio at2
Manufactured by Leica camera
Sourced in Germany, United States
The Aperio AT2 is a high-resolution digital slide scanner designed for microscopy applications. It captures digital images of tissue samples with a maximum resolution of 0.25 micrometers per pixel. The device is capable of scanning whole slide samples at various magnification levels.
Automatically generated - may contain errors
Lab products found in correlation
Imagescope, by Leica camera (4 mentions)
Recombinant human insulin, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Sigma hydrocortisone, by Merck Group (2 mentions)
Halo software, by Indica Labs (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Merck Group (2 mentions)
Bond 3 platform, by Leica camera (2 mentions)
Dpx mountant for histology, by Merck Group (2 mentions)
Aperio imagescope software, by Leica Biosystems (2 mentions)
Discovery ultra, by Roche (2 mentions)
Dab kit, by ZSGB-BIO (2 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (2 mentions)
Entkalker soft solvagreen, by Carl Roth (1 mentions)
Menzel superfrost, by Thermo Fisher Scientific (1 mentions)
Hematoxylin, by Wuhan Servicebio Technology (1 mentions)
Neutral gum, by Wuhan Servicebio Technology (1 mentions)
Rodent block m, by Biocare Medical (1 mentions)
Dab quanto chromogen substrate, by Thermo Fisher Scientific (1 mentions)
Crm 325b, by Biocare Medical (1 mentions)
68 protocols using aperio at2
1
Automated Immunohistochemistry Staining Protocol
2
Histopathological Validation of Parasite Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Histological examination was carried out in cases for which Oo was detected by molecular means to confirm infection, when tissue was still available. Tissues stored in 96% ethanol were further fixed in 10% neutral buffered formalin for 24 h, and then routinely processed into paraffin blocks with a longitudinal orientation. Sections measuring 5 μm were cut, stained with Hematoxylin-Eosin (HE), Periodic acid–Schiff (PAS) and Grocott’s methenamine silver, and examined under light microscopy. Additionally, selected slides were scanned with Leica Aperio AT2 (magnification: 20X, 0.75 Numerical Aperture, 2X optical magnifier) and used for morphometric measurements or snapshot capture with QuPath software (version 0.3.2).
3
Preparing Bone Specimens for UV-PAM Imaging
4
Tissue Processing for Histology and Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After removal from the intraoperative situs, tissue was immediately transferred to a chilled phosphate-buffered saline for transport and further processing in the laboratory.
Samples were fixed in formalin (4% PFA) for 24 h and if necessary, decalcification on an EDTA basis (Entkalker soft SOLVAGREEN®, Carl ROTH, Karlsruhe, Germany) was performed for 2–7 days. Subsequently, specimens were prepared for paraffin embedding in standard size (40 × 28 × 6.8 mm) POM histology cassettes (Kartell, Noviglio, Italy). Sections (2 µm) of paraffin-embedded samples were mounted on glass slides (Menzel SuperFrost, 76 × 26 × 1 mm, Fisher Scientific, Schwerte, Germany).
Afterwards, Hematoxylin-eosin (HE) (ethanolic eosin Y solution, Mayer’s acidic hemalum solution, Waldeck, Münster, Germany) as well as Elastica van Gieson (EvG) (picrofuchsin solution after Romeis 16th edition, Weigert’s solution I after Romeis 15th edition) stainings were performed according to the manufacturer’s protocol. Slides were covered using Pertex (Histolab products, Askim, Sweden) as the mounting medium and glass coverslips (24 × 50 mm, Engelbrecht, Edermünde, Germany).
Slides (including immunohistochemistry) were then scanned with Aperio AT2 (Leica, Wetzlar, Germany). Scanned slides were analyzed and prepared for composite figures using QuPath-0.3.2 open-source software [22 (link)].
Samples were fixed in formalin (4% PFA) for 24 h and if necessary, decalcification on an EDTA basis (Entkalker soft SOLVAGREEN®, Carl ROTH, Karlsruhe, Germany) was performed for 2–7 days. Subsequently, specimens were prepared for paraffin embedding in standard size (40 × 28 × 6.8 mm) POM histology cassettes (Kartell, Noviglio, Italy). Sections (2 µm) of paraffin-embedded samples were mounted on glass slides (Menzel SuperFrost, 76 × 26 × 1 mm, Fisher Scientific, Schwerte, Germany).
Afterwards, Hematoxylin-eosin (HE) (ethanolic eosin Y solution, Mayer’s acidic hemalum solution, Waldeck, Münster, Germany) as well as Elastica van Gieson (EvG) (picrofuchsin solution after Romeis 16th edition, Weigert’s solution I after Romeis 15th edition) stainings were performed according to the manufacturer’s protocol. Slides were covered using Pertex (Histolab products, Askim, Sweden) as the mounting medium and glass coverslips (24 × 50 mm, Engelbrecht, Edermünde, Germany).
Slides (including immunohistochemistry) were then scanned with Aperio AT2 (Leica, Wetzlar, Germany). Scanned slides were analyzed and prepared for composite figures using QuPath-0.3.2 open-source software [22 (link)].
5
Histological Analysis of Molar Resorption
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dewaxed sections were incubated with hematoxylin (Servicebio) and eosin (Servicebio); dehydrated with xylene, anhydrous alcohol, and 75% ethanol; and sealed with neutral gum (Servicebio). The cytoplasm and nucleus of all the cells turned to red and blue, respectively. The slices were scanned (Aperio AT2, Leica), and osteoclasts and tooth resorption lacuna were observed at the pressure side of the upper-left first molars.
6
Orthotopic and Metastatic Nude Mouse Models
7
Immunohistochemical Analysis of USP7 in Gastric Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gastric carcinoma tissues were acquired from the First Affiliated Hospital of Zhengzhou University. Tissue was collected according to the clinical protocol approved by the ethics review board of the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China. Before taking written consents, patients were informed about the study. Collected specimens were anonymously controlled that supported the ethical and legal standards. Deparaffinization of the slides containing histological sample were performed by xylene and different concentrations of ethanol and washed with phosphate buffer saline (PBS) three times. Then the slides were incubated with 3% hydrogen peroxidase (H2O2) at room temperature for 20 min and 5% goat serum was applied to block the nonspecific binding of antibody. Anti-USP7 antibody was used on the slides at 4 °C overnight. Following day, the slides washing were done with PBS for three times and were probed with secondary antibody for 2 h at 37 °C. Subsequently, the slides were stained with DAB and hematoxylin. Finally, the slides were dehydrated and mounted. The slides were scanned and analyzed on Aperio AT2 (Leica, Wetzlar, Germany).
8
Immunohistochemistry in Lung Tumor Models
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IHC in lung tissues of mice bearing B16F10 tumors was performed as described previously [19 (link)]. Briefly, formalin-fixed paraffin-embedded lung tissue was prepared as described for lung area measurement. We used anti-PCNA antibody (Chemicon-MAB424, 1:2500 dilution). Antibody binding was visualized using AEC (cat#3464, Dako, Glostrup, Denmark). Tissue samples were counter stained with hematoxylin according to standard methods.
IHC in lung tissues of mice bearing LLC1 tumors was performed by the Histopathology Core at the Koch Institute’s Robert A. Swanson Biotechnology Center using a Thermo Scientific LabVision 360 autostainer. Antigen retrieval was performed at 97 °C for 20 min in pH 6 citrate buffer (Abcam #3678). Slides were blocked using Rodent Block M (Biocare Medical #RBM961L). Primary antibodies were Cleaved Caspase 3 (CC3; Cell Signaling Technologies #9664 L, 1:800 dilution) and Ki-67 (Biocare Medical CRM 325B, 1:50 dilution). Secondary detection reagents were Mouse on Mouse HRP (Biocare Medical #MM620L) and Rabbit on Rodent HRP (Biocare Medical #RMR622L). After incubation with DAB Quanto Chromogen Substrate (ThermoScientific #TA-125-QHDX), slides were counterstained and scanned with Leica Aperio AT2.
IHC in lung tissues of mice bearing LLC1 tumors was performed by the Histopathology Core at the Koch Institute’s Robert A. Swanson Biotechnology Center using a Thermo Scientific LabVision 360 autostainer. Antigen retrieval was performed at 97 °C for 20 min in pH 6 citrate buffer (Abcam #3678). Slides were blocked using Rodent Block M (Biocare Medical #RBM961L). Primary antibodies were Cleaved Caspase 3 (CC3; Cell Signaling Technologies #9664 L, 1:800 dilution) and Ki-67 (Biocare Medical CRM 325B, 1:50 dilution). Secondary detection reagents were Mouse on Mouse HRP (Biocare Medical #MM620L) and Rabbit on Rodent HRP (Biocare Medical #RMR622L). After incubation with DAB Quanto Chromogen Substrate (ThermoScientific #TA-125-QHDX), slides were counterstained and scanned with Leica Aperio AT2.
9
Multicolor Immunohistochemistry for Tissue Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A 5-μm tissue section adjacent to the section used for ISH was probed for rabbit polyclonal cytokeratin 5 (KRT5, clone Poly19055, BioLegend, San Diego, CA) and mouse monoclonal pan-cytokeratin AE1/AE3 (ab27988, Abcam, Cambridge, UK) antibodies diluted to 1:200. Antigens were retrieved using sodium citrate buffer, pH 6, 100°C for 5 minutes at 5 psi. Alexafluor 555– and 488–labeled secondaries (Invitrogen, Carlsbad, CA) were used at 1:200, followed by DAPI nuclear counterstain. Slides were imaged on the Vectra Automated Multispectral Imaging System (PerkinElmer, Waltham, MA) at the Research Histology and Tissue Imaging Core at UIC. The other adjacent section was hematoxylin and eosin (H&E) stained and scanned with Aperio AT2 (Leica, Wetzlar, Germany) at the Research Histology and Tissue Imaging Core.
10
Histological Analysis of Liver Biopsies in T2DM
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Formalin-fixed paraffin-embedded liver biopsies from T2DM patients were cut with a thickness of 3 μm. After rehydration, the slides were processed for iron staining or CD68 immunohistochemistry.
Standard Perls Prussian blue staining was performed by using the Iron Stain Kit (Sigma–Aldrich). Briefly, the slides were incubated for 15 min in iron stain solution (prepared by mixing equal volumes of 4% w/v potassium ferrocyanide and 1.2 mM hydrochloric acid). Nuclear Fast Red was used as counterstain.
CD68 expression was analyzed by CD68 antibody staining (Dako; Ref-Nr.:M0876, Lot-Nr.: 00094,759; clone PG-M1) on the Ventana Benchmark Ultra (Roche) according to the manufacturer's instructions. Microscopy slides containing the tissue were preincubated with the EDTA-containing CC1 solution (Roche) for 16 min after which the slides were incubated with a 1:100 dilution of the CD68 antibody for 24 min.
The stained slides were digitally scanned with the Aperio AT2 (Leica).
Standard Perls Prussian blue staining was performed by using the Iron Stain Kit (Sigma–Aldrich). Briefly, the slides were incubated for 15 min in iron stain solution (prepared by mixing equal volumes of 4% w/v potassium ferrocyanide and 1.2 mM hydrochloric acid). Nuclear Fast Red was used as counterstain.
CD68 expression was analyzed by CD68 antibody staining (Dako; Ref-Nr.:M0876, Lot-Nr.: 00094,759; clone PG-M1) on the Ventana Benchmark Ultra (Roche) according to the manufacturer's instructions. Microscopy slides containing the tissue were preincubated with the EDTA-containing CC1 solution (Roche) for 16 min after which the slides were incubated with a 1:100 dilution of the CD68 antibody for 24 min.
The stained slides were digitally scanned with the Aperio AT2 (Leica).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!