IHC was performed as previously described (Akula et al., 2020 (link)). Primary antibodies included AP-2β (1:50, Cell Signaling, 2509S, Danvers, MA), α-smooth muscle actin (αSMA, 1:100, Sigma Aldrich, A2547, Oakville, ON), myocilin (1:200, FabGennix, MYO-101AP, Frisco, TX), endomucin (1:100, eBioscience, 14–5851-82, San Diego, CA), Prox1 (1:100, Covance, 925201, Princeton, NJ), N-cadherin (1:100, BD Transduction, 610920, San Jose, CA), and Brn3a (1:100, sc-8429, Santa Cruz, CA) (Bassett et al., 2012 (link); Martino et al., 2016 (link); Robertson et al., 2013 (link); Taiyab et al., 2021 (link)) (Table 1 ). Alexa Fluor secondary antibodies (1:200, Invitrogen, Molecular Probes, Burlington, ON) were used to detect the primary antibodies, and slides were mounted with ProLong Gold containing DAPI (4′,6-diamidino-2-phenylindole) (Thermofisher, Waltham, MA). To analyze tdTomato expression for the fate mapping experiments, frozen sections of the eyes from control and AP-2β TMR KO mice crossed with the tdTomato mouse line were used. All sections were imaged using a Leica DM6 B microscope with a bright-field or fluorescence attachment, and images were acquired using a high-resolution camera and LasX imaging software.
Endomucin
Manufactured by Thermo Fisher Scientific
Endomucin is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is a cell surface protein that serves as a marker for endothelial cells. Endomucin is commonly used in research applications to identify and study endothelial cells.
Automatically generated - may contain errors
Lab products found in correlation
Rm2235, by Leica camera (2 mentions)
Runx1, by Abcam (2 mentions)
Ti e a1 lsm 880, by Zeiss (2 mentions)
Anti mouse, by Agilent Technologies (2 mentions)
Polymer hrp anti rabbit, by Agilent Technologies (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Brn3a, by Santa Cruz Biotechnology (1 mentions)
Sc 8429, by Santa Cruz Biotechnology (1 mentions)
α smooth muscle actin α sma, by Merck Group (1 mentions)
Dm6b microscope, by Leica camera (1 mentions)
N cadherin, by BD (1 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Prox1, by Fortrea (1 mentions)
P erk thr202 tyr204, by Cell Signaling Technology (1 mentions)
Isolectin gs ib4, by Thermo Fisher Scientific (1 mentions)
P egfr, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Ab6556, by Abcam (1 mentions)
Zm 0166, by Abcam (1 mentions)
7 protocols using endomucin
1
Immunohistochemistry of Eye Tissue
2
Multicolor Immunohistochemistry for Embryo Analysis
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Embryos were isolated, fixed with 4% paraformaldehyde for 30 min to 2 h at 4 °C, embedded in paraffin, and sectioned at 5–6 μm with Leica RM2235. Sections were deparaffinized with ethanol of gradient concentration, then blocked in blocking solution (Zhongshan golden bridge) for 30 min at room temperature, followed by incubation with primary antibodies overnight at 4 °C. After 3 washes (3 min each) in PBS, sections were incubated with corresponding secondary antibodies (Zhongshan golden bridge) for 30 min at room temperature. After 3 washes in PBS, sections were stained with DendronFluor TSA (Histova, NEON 4-color IHC Kit for FFPE, NEFP450, 1:100, 20–60 s). The primary and secondary antibodies were thoroughly eluted by heating the slides in citrate buffer (pH 6.0) for 20 min at 95 °C using microwave. In a serial fashion, each antigen was labeled by distinct fluorophores. After all the antibodies were detected sequentially, the slices were finally stained with DAPI. Images were collected by confocal microscope (Nikon Ti-E A1/ ZEISS LSM 880). The primary antibodies were as follows: CD31 (BD Biosciences), CD44 (BD Biosciences), Endomucin (eBioscience), GFP (Cell Signaling), and Runx1 (Abcam).
3
Immunohistochemical Analysis of Tumor Samples
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After the mice were euthanized, the tumors were excised and fixed in formalin, embedded in paraffin, cut into 5-μm sections, dewaxed and hydrated, and stained with hematoxylin and eosin using a standard technique. The IHC staining procedures were as follows: Tissue sections were incubated for 10 min at room temperature in 3% hydrogen peroxide to block endogenous peroxidase. Slides were blocked and incubated with the following primary antibodies: anti-EGFR (Nichirei Biosciences), pEGFR (Cell Signaling Technology), pERK (Thr202/Tyr204) (Cell Signaling Technology), pAKT (Cell Signaling Technology), endomucin (eBioscience), and CD34 (Abcam). The slides were subsequently incubated with polymer-HRP anti-rabbit (Dako) or anti-mouse (Dako) secondary antibodies. Tissue staining was visualized using 4′,6-diamidino-2-phenylindole (DAPI). The slides were then counterstained with hematoxylin, dehydrated, and mounted. The coimmunofluorescence staining procedure for isolectin GS-IB4 (594) (Invitrogen) was as follows: After the same preprocessing for IHC staining as described above, slides were incubated with primary antibody against isolectin GS-IB4 (594) and subsequently incubated with the appropriate fluorescence-conjugated secondary antibodies; DAPI counterstaining was used to identify the nuclei.
4
Immunohistochemical Staining of Tissue Sections
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Sections 4 μm in thickness were de-paraffinized and rehydrated by standard protocols. Antigen retrieval was performed by heating slides in retrieval buffers pH6 in a microwave. Sections were incubated in blocking buffer (goat serum) for 1 h at room temperature and followed by incubation overnight at 4°C with primary antibodies against Ki-67 (ZSGB-Bio, ZM-0166), GFP (Abcam, ab6556, dilution 1:500), TFPI2 (Abcam, ab186747, dilution 1:100), and endomucin (Ebioscience, 14-5851, dilution 1:1,000). Sections were incubated with the secondary antibodies (rabbit (PV-6001), mouse (PV-6002), and rat (PV-6004) polymer detection system, ZSGB-Bio) for 1 h at room temperature and then stained with DAB and counterstained with hematoxylin. Staining intensity scoring was performed as previously described (37 (link)). endomucin-PAS staining was performed after staining of endomucin with DAB. Sections were incubated with 0.5% periodic acid for 15 min, rinsed in distilled water for 10 min, Schiff’s reagent for 30 min, and counterstained with hematoxylin (38 (link)).
5
Immunohistochemistry Analysis of Intestinal Tissue
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Intestines were flushed with phosphate-buffered saline (PBS) and 4% paraformaldehyde, fixed and embedded in paraffin as swiss rolls. Swiss rolls were cut into 2 µm sections and IHC/IF-stained with standard procedures using antibodies against β-catenin (Becton Dickinson, 610153, 1 : 80), BrdU (BrdU In-Situ Detection Kit, Becton Dickinson, 550803), cleaved Caspase 3 (Cell Signaling, 9661, 1 : 200), Endomucin (eBioscience, 14-5851-82, 1 : 500), GR1 (Serotec, MCA771GA, 1 : 200), Granzyme B (Abcam, ab4059, 1 : 200), IDO1 (Biolegend, 122402, 1 : 80), iNOS (Biolegend, 610431, 1 : 200), Ki67 (Novocastra, NCL-KI67-P, 1 : 1000), Lysozyme (Dako, A009902, 1 : 100), p-STAT1 (Cell Signaling, 9167S, 1 : 100), p-STAT3 (Cell Signaling, 9145, 1 : 100), STAT1 (Santa Cruz, sc-592, 1 : 500), STAT3 (Santa Cruz, sc-7179, 1 : 80), Synaptophysin (GeneTex, GTX100865, 1 : 1000), GFP (Roche, 11814460001, 1 : 1000), red fluorescent protein (RFP) (Rockland antibodies and assays, 600-401-379S, 1 : 500), CD3 (Neomarker RM9107, 1 : 100), MMP7 (Cell Signaling, 3801, 1 : 100). IHC staining on human samples was performed using antibodies against IFIT1 (Sigma Aldrich, HPA055380, 1 : 500), IDO1 (Biolegend, 122402, 1 : 100), p-STAT1 (Cell Signaling, 9167S, 1 : 100), and STAT1 (Cell Signaling, 14994, 1 : 1000).
6
Multicolor Immunohistochemistry of Embryonic Tissues
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Embryos were isolated, fixed with 4% paraformaldehyde for 30 minutes to 2 hours at 4°C, embedded in paraffin, and sectioned at 5-6 μ m with Leica RM2235. Sections were deparaffinized with ethanol of gradient concentration, then blocked in blocking solution (Zhongshan golden bridge) for 30 minutes at room temperature, followed by incubation with primary antibodies overnight at 4°C. After 3 washes (3 minutes each) in PBS, sections were incubated with corresponding secondary antibodies (Zhongshan golden bridge) for 30 minutes at room temperature. After 3 washes in PBS, sections were stained with DendronFluor TSA (Histova, NEON 4-color IHC Kit for FFPE, NEFP450, 1:100, 20-60-sec).
The primary and secondary antibodies were thoroughly eluted by heating the slides in citrate buffer (pH 6.0) for 10 minutes at 95°C using microwave. In a serial fashion, each antigen was labeled by distinct fluorophores. After all the antibodies were detected sequentially, the slices were finally stained with DAPI. Images were collected by confocal microscope (Nikon Ti-E A1/ ZEISS LSM 880). The primary antibodies were as follows: CD31 (BD Biosciences), CD44
(BD Biosciences), Endomucin (eBioscience), GFP (Cell Signaling), and Runx1 (Abcam).
The primary and secondary antibodies were thoroughly eluted by heating the slides in citrate buffer (pH 6.0) for 10 minutes at 95°C using microwave. In a serial fashion, each antigen was labeled by distinct fluorophores. After all the antibodies were detected sequentially, the slices were finally stained with DAPI. Images were collected by confocal microscope (Nikon Ti-E A1/ ZEISS LSM 880). The primary antibodies were as follows: CD31 (BD Biosciences), CD44
(BD Biosciences), Endomucin (eBioscience), GFP (Cell Signaling), and Runx1 (Abcam).
7
Immunohistochemical Analysis of Liver Samples
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Liver tissues were fixed in formalin, embedded in paraffin, cut into 5 lm sections, dewaxed, hydrated, and incubated for 10 min in 3% hydrogen peroxide to block endogenous peroxidase. Antigen retrieval was performed by washing cultured cells were with phosphate-buffered saline (PBS) and sequentially fixing with 4% paraformaldehyde. Tissue sections and cells were blocked and incubated with the following primary antibodies: GFP (Cell Signaling), BrdU (Abcam), Mac-2 (Abcam), Endomucin (eBioscience), a-smooth muscle actin (Dako), albumin (R&D Systems), CK19 (Abcam), CD14 (MBL) and CK18 (BD Biosciences). Samples were subsequently incubated with the appropriate fluorescence-conjugated secondary antibodies for co-immunofluorescence staining, while DAPI counterstaining was used to identify nuclei. Polymer-HRP anti-rabbit (Dako) or anti-mouse (Dako) secondary antibodies were used for immunohistochemical staining, and DAB reagent was employed for detection.
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