Mouse femur samples were harvested, fixed in 4% PFA, and embedded in paraffin after decalcification with 10% EDTA for 21 days. To obtain frozen sections, the samples were fixed with 4% PFA, soaked in 30% sucrose and embedded in OCT compound. A series of 10 μm sections were subjected to IHC, as well as Safranin-O/Fast green, and hematoxylin and eosin staining. For IHC staining, antigen retrieval was performed, then the sections were incubated with primary antibodies, including anti-Ki67 (1:200, 14-5698-95, Thermo Fisher Scientific, Waltham, MA, USA), anti-Osteocalcin (1:100, PB1008, Boster, Wuhan, China), anti-β-catenin (1:100, 610154, BD Biosciences, Franklin Lakes, NJ, USA), anti-Piezo1 (1:100, 15939-1-AP, Proteintech, Rosemont, IL, USA), anti-Piezo2 (1:100, 26205-1-AP, Proteintech), anti-YAP (1:100, 14074, Cell Signaling Technology, Danvers, MA, USA), anti-Endomucin (1:100, sc-65495, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-CD31 (1:50, 563607, BD Biosciences) and anti-Col2α1 (1:100, bs-11929R, Bioss Antibodies, Woburn, MA, USA). After rinsing twice, the sections were incubated with the appropriate secondary antibodies. For immunofluorescent imaging, the sections were counterstained with 4',6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St Louis, MO, USA) and imaged using confocal microscopy (Nikon A1; Nikon, Tokyo, Japan).
Anti endomucin
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-Endomucin is a laboratory reagent used for the identification and localization of endomucin, a transmembrane glycoprotein expressed on the surface of vascular endothelial cells. It can be used in various research applications that involve the study of vascular biology and endothelial cell function.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880 confocal microscope, by Zeiss (4 mentions)
Anti cleaved caspase 3 asp175, by Cell Signaling Technology (2 mentions)
Alexa fluor 488 anti rabbit, by Thermo Fisher Scientific (2 mentions)
Anti rat alexa fluor 555, by Thermo Fisher Scientific (2 mentions)
Anti ki67, by Abcam (2 mentions)
Anti ki67, by Thermo Fisher Scientific (1 mentions)
Anti piezo1, by Proteintech (1 mentions)
Anti yap, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Anti β catenin, by BD (1 mentions)
Anti cd31, by BD (1 mentions)
Hematoxylin, by Thermo Fisher Scientific (1 mentions)
Heparin, by Merck Group (1 mentions)
Dab substrate, by Vector Laboratories (1 mentions)
Alexa 647 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti cd34, by Abcam (1 mentions)
Anti ki67, by Leica (1 mentions)
Rat anti flag, by BioLegend (1 mentions)
Anti erg, by Abcam (1 mentions)
Abc elite kit, by Vector Laboratories (1 mentions)
6 protocols using anti endomucin
1
Analysis of Mouse Femur Microstructure
2
Tissue Preparation and Immunohistochemistry
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After euthanasia, mice were subjected to transcardial perfusion with PBS + heparin (Sigma-Aldrich) and 4% paraformaldehyde. Tissue samples were collected and fixed in 4% PFA in 1× PBS overnight and embedded in paraffin. Sections were stained with hematoxylin-eosin for analysis. For immunohistochemistry, antibody detection was performed using the Elite ABC kit and DAB substrate according to the manufacturer's instructions (Vector Laboratories) and counterstained with hematoxylin (Thermo Fisher Scientific). Primary antibodies used were rat anti-FLAG (Biolegend 637319), anti-CD31 (Dianova DIA-310), anti-endomucin (Santa Cruz Biotechnology sc-65495), rabbit anti-S100a6 (Novus Biologicals NBP1-89388), anti-ERG (Abcam ab133264), anti-CD34 (Abcam ab81289), anti-Ki67 (Leica Biosystems NCLKi67p), anti-cytokeratin, wide spectrum screening (Agilent Z0622), anti-CRYAB (Invitrogen PA1-16950), and mouse anti-PDGFA (Santa Cruz Biotechnology sc-9974). Alexa488-, Alexa568-, and Alexa647-conjugated secondary antibodies (Molecular Probes) were used for immunofluorescence.
3
Identification of TMEM Doorways in Tumors
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Tumor sections were deparaffinized, as described above, and stained for TMEM doorways. TMEM stain is a triple immunohistochemical stain in which 3 antibodies are applied sequentially and developed separately with different chromogens on a Dako Autostainer, as previously published51 . Briefly, we used an anti-pan-Mena antibody (BD, cat. #610693, concertation 5 µg/mL) to detect invasive tumor cells, an anti-IBA-1 antibody (Wako, cat. #019-19741, concentration 0.167 µg/mL) to detect macrophages, and an anti-endomucin (Santa Cruz, cat #sc-65495, concentration 0.67 µg/mL) to detect the blood vasculature. TMEM doorways in the E0771-primary tumor were identified manually by a pathologist.
4
Histological and Immunofluorescence Staining of Paraffin-Embedded Tissue Sections
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All tissues were fixed in 4% paraformaldehyde, paraffin-embedded, and cut into 5-μm sections for subsequent staining. For histological analysis, paraffin sections were deparaffinized, hydrated, and stained with hematoxylin and eosin (H&E). For immunohistochemical analysis, the sections were incubated in pre-warmed citrate buffer (pH 6.0) for 15 min in a low-heat microwave oven. After blocking with 10% goat serum (ZLI-9021; ZSGB-BIO) to prevent non-specific binding, the sections were incubated overnight at 4°C with primary antibodies against anti-SRSF5 (1:1500) (HPA043484; Sigma-Aldrich), anti-Ki67 (1:400) (9449S; CST), and anti-pH3 (1:500) (YP0129; Immunoway), respectively. Staining was completed using a rabbit two-step assay kit (PV-9001; ZSGB-BIO) according to the manufacturer’s instructions. Images were acquired using Pannoramic MIDI 3D HISTECH.
Immunofluorescence staining was performed in the same way as histochemical staining prior to primary antibody incubation. The primary antibodies used were anti-cTnT (1:3) (CT3; DSHB) and anti-Endomucin (1:50) (sc-65495; Santa Cruz Biotechnology). Then, the slides were washed with PBS and incubated with the fluorescent secondary antibody for 1 h at room temperature. Fluorescence nuclear staining was performed using DAPI. Fluorescent images were acquired using Pannoramic MIDI of 3D HISTECH.
Immunofluorescence staining was performed in the same way as histochemical staining prior to primary antibody incubation. The primary antibodies used were anti-cTnT (1:3) (CT3; DSHB) and anti-Endomucin (1:50) (sc-65495; Santa Cruz Biotechnology). Then, the slides were washed with PBS and incubated with the fluorescent secondary antibody for 1 h at room temperature. Fluorescence nuclear staining was performed using DAPI. Fluorescent images were acquired using Pannoramic MIDI of 3D HISTECH.
5
Immunostaining of Mouse Lung Sections
6
Immunostaining of Mouse Lung Sections
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Immunostaining of mouse lung sections was performed as described previously (Tavora et al., 2020) with the following antibodies: anti-Ki-67 (Abcam), anti-Endomucin (Santa Cruz Biotechnology), anti-cleaved Caspase3 (Asp175) (Cell Signaling Technology), anti-rabbit Alexa Fluor 488 and anti-rat Alexa Fluor 555 (Thermo Fisher Scientific). Images were acquired using a Zeiss LSM 880 confocal microscope. H&E staining of mouse lung sections were performed by Histoserv Inc.
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