Lungs from 2 week old mice were inflation fixed with 4% PFA/PBS cryoprotected in sucrose/PBS, and embedding in OCT compound (TissueTek). Thick sections (150 μm) of lung tissue were cut with a cryostat and stained using a whole mount immunofluorescence protocol (Ahnfelt-Ronne et al., 2007 (link)). Primary antibodies included goat anti-endomucin (R&D Systems) and mouse anti-alpha smooth muscle actin (Sigma), and secondary antibodies were Alexa Fluor-488 and Alexa Fluor-594. Following antibody labeling, sections were stained with DAPI and mounted on slides for imaging. A Nikon A1Rsi inverted laser confocal microscope was used to acquire 2×2 z-stack tile scans. Fluorescent images were analyzed using Imaris software and endomucin staining threshold intensities were used to generate surface representations of the pulmonary vascular network and quantify the area and volume of the lung vasculature.
Goat anti endomucin
Manufactured by R&D Systems
Sourced in United States
Goat anti-endomucin is a laboratory reagent used for the detection and analysis of endomucin, a protein expressed on the surface of endothelial cells. It can be used in various immunological techniques, such as flow cytometry, immunohistochemistry, and Western blotting, to identify and study endothelial cells.
Automatically generated - may contain errors
Lab products found in correlation
Rat anti pecam1, by BD (3 mentions)
Mouse anti alpha smooth muscle actin, by Merck Group (2 mentions)
Alexa fluor 647 donkey anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 donkey anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 donkey anti rat igg, by Thermo Fisher Scientific (2 mentions)
Goat anti foxa2, by Santa Cruz Biotechnology (2 mentions)
Goat anti pdx1, by Abcam (2 mentions)
Rat anti e cadherin, by R&D Systems (2 mentions)
Rabbit anti pax8, by Proteintech (2 mentions)
Goat anti hnf4α, by Santa Cruz Biotechnology (2 mentions)
Goat anti alb, by Fortis Life Sciences (2 mentions)
Alexa fluor 568 anti goat igg, by Thermo Fisher Scientific (2 mentions)
A1rsi inverted laser confocal microscope, by Nikon (1 mentions)
Af1623, by R&D Systems (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Af305 na, by R&D Systems (1 mentions)
Af4666, by R&D Systems (1 mentions)
Alexa fluor 488 donkey anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions)
Goat anti ephb4, by R&D Systems (1 mentions)
Axio imager a2 microscope, by Zeiss (1 mentions)
5 protocols using goat anti endomucin
1
Quantitative Assessment of Lung Vasculature
2
Immunofluorescence Staining of Murine Placental Tissue
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Placentas at 18.5 dpc were collected and fixed overnight in 4% PFA at 4°C. The tissues were then processed for paraffin embedding. Sections of size 5 μm thickness were sliced,
deparaffinized, and rehydrated. Antigen retrieval was performed by boiling the sections for 20 min in a citrate-based antigen-unmasking solution. Non-specific binding was blocked using 4%
normal horse serum for 30 min.
The following primary antibodies were used: Rabbit anti-Nrk (Merck, HPA017238, 1:100), goat anti-endomucin (R&D Systems, MN, USA, AF4666, 1:200), goat anti-proliferin (R&D Systems,
AF1623, 1:200), and goat anti-IGF1r (R&D Systems, AF305-NA, 1:200), which were incubated overnight at 4°C. The secondary antibodies used were Alexa Fluor® 488 donkey anti-rabbit IgG
H&L (Jackson ImmunoResearch, 711-545-152, 1:500) and Alexa Fluor® 594 donkey anti-goat IgG H&L (Abcam, Cambridge, UK, ab150132, 1:500), which were incubated for 1 h at 20–25°C.
Counterstaining and mounting were performed using VECTASHIELD® with DAPI (Vector Laboratories, H-1200).
deparaffinized, and rehydrated. Antigen retrieval was performed by boiling the sections for 20 min in a citrate-based antigen-unmasking solution. Non-specific binding was blocked using 4%
normal horse serum for 30 min.
The following primary antibodies were used: Rabbit anti-Nrk (Merck, HPA017238, 1:100), goat anti-endomucin (R&D Systems, MN, USA, AF4666, 1:200), goat anti-proliferin (R&D Systems,
AF1623, 1:200), and goat anti-IGF1r (R&D Systems, AF305-NA, 1:200), which were incubated overnight at 4°C. The secondary antibodies used were Alexa Fluor® 488 donkey anti-rabbit IgG
H&L (Jackson ImmunoResearch, 711-545-152, 1:500) and Alexa Fluor® 594 donkey anti-goat IgG H&L (Abcam, Cambridge, UK, ab150132, 1:500), which were incubated for 1 h at 20–25°C.
Counterstaining and mounting were performed using VECTASHIELD® with DAPI (Vector Laboratories, H-1200).
3
Whole-Mount Immunohistochemistry for Tissue Analysis
4
Tissue Fixation and Immunostaining Protocols
5
Whole-Mount Immunohistochemistry for Tissue Analysis
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Whole-mount immunohistochemistry was performed using a modification of
the protocol fromAhnfelt-Rønne et al.
(2007) as previously described (Havrilak and Shannon, 2015b (link)). Optical sections were captured on a
Nikon AiRsi inverted laser microscope, and 3D images were created by a composite
of z-stacks using Bitplane Imaris software to process the fluorescent images. To
calculate the volume of tissue stained for ALB, we created isosurfaces using
smoothing thresholds and intensity values that allowed filtering out antibody
aggregates or any non-specific staining.
The primary antibodies used were: rabbit anti-NKX2-1 (1:3000, Seven
Hills Bioreagents), guinea pig anti-NKX2-1 (1:500, Seven Hills Bioreagents),
goat anti-endomucin (1:500, R & D Systems), rat anti-E-cadherin (1:2000,
R & D Systems), rabbit anti-PAX8 (1:500, Protein Tech), rat anti-PECAM-1
(1:500, BD Pharmigen), goat anti-HNF4α (1:200, Santa Cruz), goat
anti-PDX1 (1:5000, Abcam), goat anti-ALB (1:1000, Bethyl Laboratories), goat
anti-FOXA2 (1:500, Santa Cruz), and rat anti-LIV2 (1:200, MBL). Secondary
antibodies used were: Alexa Fluor 647 donkey anti-rabbit IgG, Alexa Fluor 568
anti-goat IgG, Alexa Fluor 488 donkey anti-rat IgG, Alexa Fluor 488 donkey
antimouse IgG (1:500, all from Life Technologies).
the protocol from
(2007)
Nikon AiRsi inverted laser microscope, and 3D images were created by a composite
of z-stacks using Bitplane Imaris software to process the fluorescent images. To
calculate the volume of tissue stained for ALB, we created isosurfaces using
smoothing thresholds and intensity values that allowed filtering out antibody
aggregates or any non-specific staining.
The primary antibodies used were: rabbit anti-NKX2-1 (1:3000, Seven
Hills Bioreagents), guinea pig anti-NKX2-1 (1:500, Seven Hills Bioreagents),
goat anti-endomucin (1:500, R & D Systems), rat anti-E-cadherin (1:2000,
R & D Systems), rabbit anti-PAX8 (1:500, Protein Tech), rat anti-PECAM-1
(1:500, BD Pharmigen), goat anti-HNF4α (1:200, Santa Cruz), goat
anti-PDX1 (1:5000, Abcam), goat anti-ALB (1:1000, Bethyl Laboratories), goat
anti-FOXA2 (1:500, Santa Cruz), and rat anti-LIV2 (1:200, MBL). Secondary
antibodies used were: Alexa Fluor 647 donkey anti-rabbit IgG, Alexa Fluor 568
anti-goat IgG, Alexa Fluor 488 donkey anti-rat IgG, Alexa Fluor 488 donkey
antimouse IgG (1:500, all from Life Technologies).
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