Formalin fixed brain hemispheres were decontaminated in formic acid for 60 min to eliminate prion infectivity. After additional fixation in formalin, the tissue was paraffin embedded. Paraffin sections (3 μm) were stained with H&E. Immunohistochemical stainings for GFAP (1:13000 DAKO) and IBA1 (1:1000) were performed using standard methods. For SAF84 immunostains, sections were incubated in 98% formic acid for an additional 6 min after deparaffinization and washed in distilled water for 30 min. Sections were treated with citrate buffer (pH 6.0) for 3 min at 100°C. After adapting to room temperature, sections were incubated in Ventana buffer, and stained with the NEXEX immunohistochemistry robot (Ventana Instruments) using an iVIEW DAB Detection Kit (Ventana). After incubation with protease 2 (Ventana) for 16 min, sections were incubated with anti-PrP SAF-84 (SPI-Bio, A03208, 1:200) for 32 min and counterstained with hematoxylin.
Protease 2
Manufactured by Roche
Sourced in United States
Protease 2 is a laboratory instrument designed to facilitate the study and analysis of proteins. It is a versatile tool used for the detection, separation, and characterization of various protease enzymes. The core function of Protease 2 is to enable researchers to isolate and identify specific proteases, which play a crucial role in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Iview dab detection kit, by Roche (3 mentions)
Ventana discovery ultra platform, by Roche (2 mentions)
Prolong gold and dapi, by Thermo Fisher Scientific (2 mentions)
Q dot linked fluorescent secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (2 mentions)
Nanozoomer digital pathology system ndpview, by Hamamatsu Photonics (2 mentions)
Benchmark xt, by Roche (1 mentions)
Eber probe, by Roche (1 mentions)
Ventana ish iview blue detection kit, by Roche (1 mentions)
Discovery ultra, by Roche (1 mentions)
Discovery xt, by Roche (1 mentions)
Discovery cc1, by Roche (1 mentions)
Ki 67 primary antibodies, by Abcam (1 mentions)
7 protocols using protease 2
1
Prion Decontamination and Immunohistochemical Analysis of Formalin-Fixed Brain Tissue
2
Immunostaining of EGFRvIII-expressing GBM Xenograft
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An EGFRvIII-expressing human GBM (GBM39) was propagated as a xenograft40 (link) and kindly provided by C. David James (Department of Neurological Surgery, University of California San Francisco). Harvested tumor tissue was formalin-fixed, paraffin-embedded, and cut into 4 µm sections for mounting on positively-charged slides. Antigen retrieval was performed using protease 2 (Ventana). Sections were immunostained with primary antibodies targeting phospho-Tyr-845 (1:150; Abcam) and human uPAR (1:75; Dako) for 1 h at 37°C using the Ventana Discovery Ultra Platform. Q-dot-linked fluorescent secondary antibodies (1:150; Invitrogen) were added for 1 h. The slides were rinsed and cover-slipped with Prolong Gold and DAPI (Invitrogen). Slides were visualized on a Zeiss Axio Imager2 using Cambridge Research Instruments Nuance Multispectral Imaging System software to capture images and visualize individual fluorophore spectra free from auto-fluorescence noise. In control experiments, phospho-epitope labeling was validated using protein phosphatase treatment, which eliminated signal.
3
Automated EBER-ISH for EBV Detection
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EBER-ISH was performed on the tissue sections using the BenchMark XT automated slide stainer (Ventana Medical Systems, Tucson, AZ, USA). The sections were treated with protease 2 (catalog no. 780-4147; Ventana Medical Systems) and labeled with an EBER probe (catalog no. 780-2842; Ventana Medical Systems) for 2 h. Hybridization products were visualized with Ventana ISH iView Blue Detection Kit (Ventana Medical Systems) according to the manufacturer’s instructions. Dark blue dots at the site of hybridization (nucleus) was regarded as positive for EBV.
4
Immunohistochemical Staining of CNS Tissues
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Stainings were performed on 2 µm sections from formalin fixed, formic acid treated, paraffin embedded tissues. After deparaffinization through graded alcohols, heat-induced antigen retrieval was performed in EDTA-based buffer CC1 and stainings were performed on a NEXES immunohistochemistry robot (Ventana instruments) using the following antibodies: Iba1 (1:1000, Wako); GFAP (1:13000, Dako); SAF84 (1:200, SPI bio). Only for the latter staining, antibody incubation was preceded by incubation with protease 2 (Ventana). Immunoreactivity was visualized using an IVIEW DAB Detection Kit (Ventana). Haematoxylin and eosin staining was performed according to standard procedures. Slides were scanned with NanoZoomer and images were visualized using the NanoZoomer Digital Pathology System (NDPview, Hamamatsu Photonics).
5
Immunostaining of EGFRvIII-expressing GBM Xenograft
6
Quantitative Histological Analysis of Brain Tissue
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Histological analyses were performed on 2-μm thick sections from formalin fixed, formic acid treated, paraffin embedded brain tissues, as previously described[32 (link)]. Sections were subjected to deparaffinization through graded alcohols, followed by heat-induced antigen retrieval performed in EDTA-based buffer CC1 (Ventana). Stainings were performed on a NEXES immunohistochemistry robot (Ventana instruments) with the following antibodies: Iba1 (1:1000, Wako); GFAP (1:13000, Dako); SAF84 (1:200, SPI bio). For the latter staining, incubation with protease 2 (Ventana) was performed before antibody incubation. Immunoreactivity was visualized using an IVIEW DAB Detection Kit (Ventana). Haematoxylin and eosin stainings were performed according to standard procedures. Slides were scanned with NanoZoomer and images were acquired using the NanoZoomer Digital Pathology System (NDPview, Hamamatsu Photonics). High magnification pictures were quantified with an in-house developed python script by counting the number of HRP pixels, which were defined as pixels for which the following condition in RGB colour space holds true: R > G > B.
7
Immunohistochemistry Protocol for CD13 and Ki-67
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