To measure SSTR2 mRNA expression levels, snap-frozen tissue was cryosectioned, mixed with lysis buffer and shortly centrifuged. The supernatant was used to determine mRNA expression levels as previously described [6 (link)]. For SSTR2 immunohistochemistry, automated IHC, using the Ventana Discovery Ultra (Ventana Medical Systems Inc., Almere, The Netherlands), was used. Formalin-fixed paraffin-embedded sections (4 µm) were stained for SSTR2A, using a universal chromomap DAB detection Kit (#760-159, Ventana). In brief, following deparaffinization and heat-induced antigen retrieval with CC1 (#950-500, Ventana) for 92 min, the tissue samples were incubated with the SSTR2A antibody (Biotrend, Maastricht, The Netherlands, rabbit polyclonal, lot number D19803) in a 1:25 dilution for 60 min at 37 °C. Subsequently, tissue sections were incubated with secondary antibody omnimap anti-rabbit HRP (#760-4311, Ventana) for 20 min, followed by DAB detection. Incubation was followed by hematoxylin II counter-staining (#790-2208, Ventana) for eight minutes, after which tissue sections were exposed to a blue coloring reagent (#790-2037, Ventana) for eight minutes, according to the manufacturer’s instructions. Stained sections were imaged and quantified by using the method described for the in vitro studies.
Omnimap anti rabbit hrp
Manufactured by Roche
The OmniMap anti-rabbit HRP is a laboratory equipment product. It is a horseradish peroxidase (HRP) conjugated secondary antibody that specifically binds to rabbit primary antibodies. The core function of this product is to enable the detection and visualization of target proteins in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ventana discovery ultra, by Roche (3 mentions)
Chromomap dab detection kit, by Roche (3 mentions)
Discovery ultra, by Roche (3 mentions)
Discovery cc1, by Roche (2 mentions)
Omnimap anti goat hrp, by Roche (2 mentions)
Discovery chromomap dab detection kit, by Roche (2 mentions)
Discovery ultra processor, by Roche (2 mentions)
Blue coloring reagent, by Roche (1 mentions)
Discovery chromomap dab kit, by Roche (1 mentions)
Cp229c, by Biocare Medical (1 mentions)
Superfrost plus, by Thermo Fisher Scientific (1 mentions)
Eclipse ni microscope, by Nikon (1 mentions)
Peroxidase block, by Agilent Technologies (1 mentions)
Discovery xt, by Roche (1 mentions)
Nbp1 90156, by Novus Biologicals (1 mentions)
Chromomap dab, by Roche (1 mentions)
Aperio versa, by Leica (1 mentions)
Bluing reagent, by Roche (1 mentions)
Red 610 kit, by Roche (1 mentions)
Dab map detection kit, by Roche (1 mentions)
10 protocols using omnimap anti rabbit hrp
1
Quantifying SSTR2 mRNA and Protein Levels
2
Apoptosis Quantification in Xenografts
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Xenografts were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 4 μM, stained with hematoxylin and eosin and reviewed by light microscopy using an upright Nikon Eclipse Ni microscope (Nikon Instruments, Inc.). Immunohistochemistry was performed on 4 μm thick tissue sections mounted on positively charged glass slides (Superfrost Plus; Thermo Fisher Scientific, Waltham, MA), and dried at 60°C for 20 min. The procedures for immunohistochemistry were performed using a Ventana DISCOVERY ULTRA autostainer (Roche). Analysis of cleaved caspase 3 IHC . Immunohistochemistry for cleaved caspase 3 (BioCare medical, CP229C, 1:500) was performed using a Ventana DISCOVERY ULTRA automated stainer (Ventana Medical Systems, Inc., Tucson, AZ). Heat-induced epitope retrieval (HIER) was carried out for 32 min at 37°C using Cell conditioning media 1 (CC1, 950-124, Ventana Medical Systems, Inc, Tucson, AZ) followed by incubation with the primary antibody for 60 min and then visualization with OmniMap anti-rabbit HRP (760–4311, Ventana Medical Systems, Inc, Tucson, AZ) and the DISCOVERY ChromoMap DAB kit (760-159, Ventana Medical Systems, Inc, Tucson, AZ). Positive cells in different views under microscope were counted as individuals for unpaired Student’s t test.
3
Immunohistochemical Analysis of Liver Macrophages and T Cells
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For immunohistochemistry analysis, mice were euthanized 24 h after the last treatment injection and livers were fixed in 4% neutral-buffered formalin (Formafix). Immunohistochemistry was performed using the horseradish peroxidase (HRP) method to identify macrophages (Iba1+) and T cells (CD3+). Briefly, after deparaffination, sections underwent antigen retrieval in citrate buffer with a pH 6.0 (Iba1) or Tris/EDTA buffer (pH 9) for 20 min at 98 °C (CD3), followed by incubation with the primary antibodies (Iba1, polyclonal antibody, rabbit anti-human, WAKO, 1:750; CD3, monoclonal antibody rabbit anti-mouse, 1:900, Ventana; both antibodies diluted in dilution buffer, Agilent Dako). This was followed by the blocking of endogenous peroxidase (peroxidase block, Agilent Dako) for 10 min at RT and incubation with the appropriate secondary antibodies/detection systems (Iba1: HRP EnVision+ rabbit, Agilent Dako; CD3: OmniMap anti-rabbit HRP, Ventana), all in an autostainer. Sections were subsequently counterstained with hematoxylin. A lymph node from a normal mouse served as a positive control (Laboratory for Animal Model Pathology, University of Zurich, Switzerland).
4
Quantification of ANXA10 Expression in Colorectal Serrated Crypts
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IHC of all HPs and SSPs was performed on 4-μm sections of formalin-fixed, paraffin-embedded tissues using a Discovery XT automated stainer (Ventana Medical Systems, Tucson, AZ). Rabbit polyclonal, affinity purified anti-ANXA10 (NBP1–90156, Novus, Littleton, CO; 1:250) was applied for 1 h at 37 °C. A secondary antibody (OmniMap anti-rabbit HRP; Ventana Medical Systems) was applied for 32 min at 37 °C. The chromogenic substrate (ChromoMapDAB; Ventana Medical Systems) was applied for 8 min at 37 °C. Slides were counterstained and visualized by a light microscope. The percentage of serrated crypts positively staining for ANXA10 were scored according to the following scoring system:
0—No crypts stained positive for ANXA10;
1—<5% of crypts stained positive for ANXA10;
2—5–25% of crypts stained positive for ANXA10;
3—26–50% of crypts stained positive for ANXA10;
4—51–75% of crypts stained positive for ANXA10; and
5—76–100% of crypts stained positive for ANXA10.
0—No crypts stained positive for ANXA10;
1—<5% of crypts stained positive for ANXA10;
2—5–25% of crypts stained positive for ANXA10;
3—26–50% of crypts stained positive for ANXA10;
4—51–75% of crypts stained positive for ANXA10; and
5—76–100% of crypts stained positive for ANXA10.
5
Multiplex IHC Staining and Analysis
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All samples for IHC were FFPE into blocks. FFPE blocks were sectioned at 4 mm. IHC was run on a Ventana Discovery Ultra. Primary antibodies used are listed in Supplementary Methods. The Chromomap DAB detection kit (Ventana; 760-159) and OmniMap anti-Rabbit HRP (Ventana; 760-4311) were used for rabbit primary antibodies. DABMap detection kit (Ventana; No. 760-124) and biotinylated antirat secondary antibody-mouse adsorbed (Vector Labs; No. BA9401)were used for rat primary antibodies. All chromogenic staining was done using DAB as a substrate and counterstained using Hematoxylin II (Ventana; No. 790-2208) and Bluing Reagent (Ventana; No. 760-2037). For fluorescent multiplexing, Ki67, granzyme B, and CD8 primary antibodies were sequentially stained, with a CC2 denaturation step in between. The following substrates were used for a fluorescence Red 610 kit (Ventana; No. 760-245), DCC kit (Ventana; No. 760-240), and FAM (Ventana; No. 760-243), and the counterstain used was QD DAPI (Ventana; No. 760-4196). Chromogenic slides were scanned using an Aperio AT2 (Leica), and fluorescent slides were scanned with an Aperio Versa (Leica). Image analysis was performed on the HALO platform (Indica Labs).
6
Immunohistochemical Profiling of TMPRSS2, AR, and ACE2
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Immunohistochemistry staining was performed using the Discovery ULTRA automated stainer from Roche Diagnostics (Indianapolis, IN). In brief, antigen retrieval was performed using a tris/borate/EDTA buffer (Discovery CC1, 06414575001; Roche), pH 8.0 to 8.5, at 95° C for 32 minutes. For AR staining only, 64 minutes of antigen retrieval time was applied. The slides were then incubated with primary antibodies for 1 hour at room temperature with the following dilutions: TMPRSS2 (ab92323), 1:3000; TMPRSS2 (ab214462), 1:200; Androgen Receptor (ab133273), 1:100; ACE2 (R&D Systems, #AF933), 1:400. The antibodies were visualized using the OmniMap anti-Rabbit HRP (05269679001; Roche), and OmniMap anti-Goat HRP (06607233001; Roche) in conjunction with the ChromoMap DAB detection kit (05266645001; Roche). Lastly, the slides were counterstained with hematoxylin and bluing. The specificity of each antibody was first tested on appropriate control tissues before proceeding to staining of the lung sections.
7
Histological Evaluation of Lung Inflammation
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Lungs used for histology and IHC examinations were slowly perfused with a fixative solution of 4% paraformaldehyde. Sections of 4 μm in thickness were stained with hematoxylin and eosin and evaluated under light microscopy using a Nikon Eclipse Ti microscope and ImageJ software (ImageJ 1.40 g; W. Rasband). They were analyzed by three independent investigators blinded to the treatment. Inflammation and damage were scored as follows: normal lung (0), hemorrhage (0‐1), peribronchial infiltration (0‐1), pulmonary edema (0‐2), alveolar hyperplasia (0‐3), and intra‐alveolar infiltration (0‐3). The total histopathology score was the sum of the scores of each variable, which were also separately evaluated. For IHC, a polyclonal rabbit anti‐human fibrinogen/fibrin antibody (1:6000; Abcam) followed by OmniMap anti‐Rabbit HRP (Roche, Merck Millipore) were used. Isotype and negative control were performed with Rabbit IgG polyclonal antibody (Abcam) and the omission of the primary antibody, respectively. Entire sections were digitized with NanoZoomer (Hamamatsu photonics) and fibrinogen/fibrin positive areas were quantified by ImageJ software (ImageJ 1.40g; W. Rasband) and expressed as a percentage of the total lung.
8
IHC Analysis of Ki-67 and Cleaved Caspase-3
9
Immunohistochemical Evaluation of SARS-CoV-2 Host Factors
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Immunohistochemistry staining was performed using the Discovery ULTRA automated stainer from Roche Diagnostics (Indianapolis, IN). In brief, antigen retrieval was performed using a tris/borate/EDTA buffer (Discovery CC1, 06414575001; Roche), pH 8.0 to 8.5, at 95 °C for 32 min. For AR staining only, 64 min of antigen retrieval time was applied. The slides were then incubated with primary antibodies for 1 h at room temperature with the following dilutions: TMPRSS2 (ab92323), 1:3000; TMPRSS2 (ab214462), 1:200; Androgen Receptor (ab133273), 1:100; ACE2 (R&D Systems, #AF933), 1:400. The antibodies were visualized using the OmniMap anti-Rabbit HRP (05269679001; Roche), and OmniMap anti-Goat HRP (06607233001; Roche) in conjunction with the ChromoMap DAB detection kit (05266645001; Roche). Lastly, the slides were counterstained with hematoxylin and bluing. The specificity of each antibody was first tested on appropriate control tissues (Fig. S3 ) before proceeding to staining of the lung sections. Controls without primary antibody were included in all experiments (Fig. S4 ).
10
Quantification of Proliferation and Apoptosis in Xenograft Tumors
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Immunohistochemistry (IHC) for Ki-67 and cleaved caspase 3 was performed in Molecular Cytology Core Facility of Memorial Sloan Kettering Cancer Center using a Discovery Ultra processor (Ventana Medical Systems). The Ki-67 probe (rabbit monoclonal Ki-67 antibody, Cell Signaling, #9027) was used at 1 ug/ml concentration and the cleaved caspase 3 probe (rabbit polyclonal cleaved caspase-3 antibody, Cell Signaling, #9661) was used at 0.2ug/ml concentration. Incubation with the primary antibody was done for 4 hours, followed by 60 minutes incubation with an OmniMap anti-rabbit HRP (Roche Diagnostics Cat#760-4311) secondary antibody. Discovery ChromoMap DAB detection kit (Roche Diagnostics) was used according to the manufacturer instructions. LK-2 xenograft tumors treated with vehicle, everolimus, and TAK-228 were tested. Tumors were harvested at the end of treatment (~ day 23). Slides were reviewed and scored by two pathologists, and differences assessed for statistical significance using GraphPad Prism 9 (unpaired t-tests).
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