Tissue sections were treated with xylene and descending alcohol and stained with picrosirius red (Sigma-Aldrich) at room temperature for 1 h. Tissue sections were then washed with 1% acidified water and sealed by a coverslip using the Surgipath micromount (Leica).
Surgipath micromount
Manufactured by Leica
Sourced in Italy, Germany
The Surgipath Micromount is a laboratory equipment product designed for the preparation of tissue samples for microscopic examination. It provides a secure and stable mounting platform for the specimen, facilitating its analysis under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Picrosirius red, by Merck Group (1 mentions)
Ab40790, by Abcam (1 mentions)
Ab9391, by Abcam (1 mentions)
Ir750, by Agilent Technologies (1 mentions)
Abc kit, by Vector Laboratories (1 mentions)
Hematoxylin, by Leica (1 mentions)
Cc1 buffer, by Roche (1 mentions)
Biotinylated goat anti rabbit igg, by Vector Laboratories (1 mentions)
Panoramic midi 2 scanner, by 3DHISTECH (1 mentions)
Discovery xt processor, by Roche (1 mentions)
Ezprep buffer, by Roche (1 mentions)
Avidin biotin blocking, by Roche (1 mentions)
Nis elements d 3, by Nikon (1 mentions)
Eclipse 50i, by Nikon (1 mentions)
Tween 20, by Merck Group (1 mentions)
Vecstain elite abc kit, by Vector Laboratories (1 mentions)
Bs 0782r, by Bioss Antibodies (1 mentions)
Vector sg peroxidase substrate kit, by Vector Laboratories (1 mentions)
Nuclear fast red, by Vector Laboratories (1 mentions)
Xylene, by Merck Group (1 mentions)
6 protocols using surgipath micromount
1
Picrosirius Red Staining Protocol
2
Immunohistochemical Analysis of RAS Components in Meningioma
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Four micrometer thick formalin-fixed paraffin-embedded sections of WHO grade I MG samples from 11 patients were subjected to 3,3-diaminobenzidine (DAB) IHC staining for (pro)renin receptor (PRR) (1:2000; cat# ab40790, Abcam, Cambridge, UK), ACE (1:100; cat# MCA2054, AbD Serotec, Kidlington, UK), ATIIR1 (1:30; cat# ab9391, Abcam and ATIIR2 (1:2000; cat# NBP1-77368, Novus Biologicals, LLC, Littleton, CO, USA). Surgipath Micromount (Leica) was used to mount all the slides. Staining of MG sections with a mouse (ready-to-use; cat# IR750, Dako, Copenhagen, Denmark) and rabbit (ready-to-use; cat# IR600, Dako) primary antibody isotype control combination was performed as an appropriate negative control, as previously described (24 (link)).
Two of the MG samples subjected to DAB IHC staining underwent immunofluorescence (IF) IHC staining using combinations of CD34 (ready-to-use; cat# PA0212, Leica), ERG (ready-to-use; cat# EP111, Cell Marque, Rocklin, CA, USA) (25 (link)), and OCT4 (1:30; cat# MRQ-10, Cell Marque) that highlighted the endothelium and stem cells on the microvessels, respectively (13 (link)). Appropriate positive human control tissues included placenta for PRR, liver for ACE and ATIIR1, and kidney for ATIIR2.
Two of the MG samples subjected to DAB IHC staining underwent immunofluorescence (IF) IHC staining using combinations of CD34 (ready-to-use; cat# PA0212, Leica), ERG (ready-to-use; cat# EP111, Cell Marque, Rocklin, CA, USA) (25 (link)), and OCT4 (1:30; cat# MRQ-10, Cell Marque) that highlighted the endothelium and stem cells on the microvessels, respectively (13 (link)). Appropriate positive human control tissues included placenta for PRR, liver for ACE and ATIIR1, and kidney for ATIIR2.
3
Immunohistochemical Analysis of Tumor Markers
4
Immunohistochemical Analysis of IL-6 and TNF-α
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The sections were permeabilized with a 0.1% Tween 20 (Sigma-Aldrich, Milan, Italy) solution in PBS, endogenous peroxidase was blocked with 1% hydrogen peroxide (Marco Viti Pharmaceutical, Sandrigo, Vicenza, Italy), and non-specific sites were saturated with the blocking buffer Vecstain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA). Sections were then incubated with the primary antibody at the concentrations 1:250 for IL-6 (bs-0782R, Bioss Inc, Woburn, Massachusetts, USA) and 1:200 for TNFalfa (ab1793, abcam, San Francisco, USA) overnight at 4°C.
After 24 h, the incubation with the appropriate secondary antibody Vecstain Elite ABC kit (Vector Laboratories) was performed, followed by signal amplification and detection with Vector SG Peroxidase substrate kit (Vector) and the Nuclear Fast Red (Vector Laboratories). Finally, after dehydration and a passage in xylene, the slides were mounted with Surgipath® Micromount (Leica Biosystem, Buccinasco, Milan, Italy) and observed with the optical microscope ECLIPSE 50i (Nikon). Two samples per groups were analyzed at the cochleostomy and mid-modiolar region (about 15 sections each). The images were acquired with Nis Elements D 3.2 software (Nikon).
After 24 h, the incubation with the appropriate secondary antibody Vecstain Elite ABC kit (Vector Laboratories) was performed, followed by signal amplification and detection with Vector SG Peroxidase substrate kit (Vector) and the Nuclear Fast Red (Vector Laboratories). Finally, after dehydration and a passage in xylene, the slides were mounted with Surgipath® Micromount (Leica Biosystem, Buccinasco, Milan, Italy) and observed with the optical microscope ECLIPSE 50i (Nikon). Two samples per groups were analyzed at the cochleostomy and mid-modiolar region (about 15 sections each). The images were acquired with Nis Elements D 3.2 software (Nikon).
5
Immunohistochemical Tissue Staining Protocol
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Skin samples for tissue staining were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4 °C for 1 day and then embedded in paraffin. The tissues were sectioned to a 6-μm thickness. Paraffin was removed by xylene (Sigma-Aldrich). Sections were soaked in descending alcohol. The tissue sections were boiled in sodium citrate (pH 6) for antigen retrieval, treated with 3.5% H2O2 (Sigma-Aldrich) in methanol, blocked using 5% goat serum for 1 h at room temperature, treated with the primary antibody overnight at 4 °C, and then incubated with the secondary antibody conjugated with either horseradish peroxidase (Invitrogen) or fluorescent dyes (Alexa Fluor 488- or 546-conjugated secondary antibodies) (Invitrogen) for 2 h at room temperature. For immunofluorescence staining, 4',6-diamidino-2-phenylindole (DAPI) was used as a nuclear counterstain. For immunohistochemistry, samples were further treated with 3,3'-diaminobenzidine (Dako, Glostrup Kommune, Denmark) and were then counterstained with hematoxylin (Millipore). The slides for immunohistochemistry were sealed with a coverslip using the Surgipath micromount (Leica Biosystems, Wetzlar, Germany). The slides for immunofluorescence staining were sealed using Flouromount-G (Invitrogen).
6
Immunohistochemical Analysis of Apoptosis and Proliferation in Xenograft Tumors
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Antibody staining was performed on histological sections of 4% paraformaldehyde-fixed OE19 tumor xenografts. Prior to immunohistochemical staining, endogenous peroxidase activity was quenched with 3% (v/v) hydrogen peroxide for 30 min. Antigen retrieval was enhanced by microwaving in 10 mM sodium citrate, pH 6.0. Nonspecific binding was blocked with 3% normal horse serum in PBS for 30 minutes. Cleaved caspase-3 staining was performed with an anti–cleaved caspase-3 primary antibody (1:1,000 dilution; catalog 9661; Cell Signaling Technology) that specifically recognizes the large fragment (17 kDa) of the active protein but not full-length caspase-3. Similarly, Ki-67 immunostaining was performed with a polyclonal anti–Ki-67 (1:200 dilution; ab15580; Abcam) antibody. Staining was detected using an avidin-biotin horseradish peroxidase system (Vectastain Universal Elite ABC kit, Cat. PK-6200, Vector Laboratories, Burlingame, CA), with positive cells staining brown using diaminobenzidine chromogen and hydrogen peroxide substrate (two-component DAB pack HK542-XAK, BioGenex, San Ramon, CA). Slides were counterstained with modified Harris hematoxylin. Tissue sections were dehydrated through graded ethanol and mixed xylenes and mounted onto coverslips with mounting medium (Surgipath Micromount, Leica Biosystems, Richmond, IL).
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