Lungs were harvested and fixed as previously described (Frank et al., 2019 (link)). They were then washed, dehydrated, embedded in paraffin, and 6-µm sections were obtained. IHC was performed on E15.5, E17.5 and E18.5 sections. In brief, the sections were deparaffinized and rehydrated followed by antigen retrieval (Reveal Decloaker, MSPP-RV1000M, Biocare Medical). The sections were incubated with 3% H2O2 for 15 min to quench endogenous peroxidases, blocked with 5% donkey serum, and, finally, incubated with primary antibody (rabbit anti-VWF, 1:250, F3520, Sigma-Aldrich; goat anti-EMCN, 1:200, AF4666, R&D Systems; mouse anti-ACTA2, 1:500, A5228, Sigma-Aldrich; rabbit anti-RFP, 1:50, 600-401-379, Rockland Immunochemicals) in 0.1% PBST overnight at 4°C. The presence of relevant proteins was visualized using Alexa Fluor secondary antibodies (donkey anti-mouse Alexa Fluor 488, 1:250, A-21202, Sigma-Aldrich; donkey anti-rabbit Alexa Fluor 555, A-31572, Thermo Fisher Scientific; donkey anti-goat Alexa Fluor 647 1:250, A-21447, Thermo Fisher Scientific).
Af4666
Manufactured by R&D Systems
Sourced in United States
AF4666 is a laboratory instrument designed for the separation and analysis of macromolecules, such as proteins, nucleic acids, and other high-molecular-weight compounds. The core function of this product is to perform analytical fractionation using the technique of Asymmetric Flow Field-Flow Fractionation (AF4).
Automatically generated - may contain errors
Lab products found in correlation
A21447, by Thermo Fisher Scientific (2 mentions)
F3520, by Merck Group (2 mentions)
A5228, by Merck Group (1 mentions)
A21202, by Merck Group (1 mentions)
A31572, by Thermo Fisher Scientific (1 mentions)
A31573, by Thermo Fisher Scientific (1 mentions)
A21208, by Thermo Fisher Scientific (1 mentions)
Rm 9106, by Thermo Fisher Scientific (1 mentions)
A11055, by Thermo Fisher Scientific (1 mentions)
Af1623, by R&D Systems (1 mentions)
Af305 na, by R&D Systems (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Goat anti endomucin, by R&D Systems (1 mentions)
Alexa fluor 488 donkey anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions)
Dmi8 confocal microscope, by Leica (1 mentions)
A21202, by Thermo Fisher Scientific (1 mentions)
4 protocols using af4666
1
Immunohistochemistry of Embryonic Lung Vasculature
2
Multicolor Immunofluorescence for Ki67, CD45, and Endomucin
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Slides containing cytospin-isolated cells were fixed and permeabilized with 3.7% formaldehyde and 0.1% triton for 20 min at room temperature (RT). After blocking with 5% BSA in PBS for 1 h, slides were incubated with anti-Ki67 (1:200, thermofisher-RM-9106), CD45 (1:200, Invitrogen-16-0451), and endomucin antibodies (1:200, R&D-AF4666) overnight at 4 °C. Slides were washed with PBST buffer followed by incubation with donkey anti-rat (1:500, Invitrogen A-21208), donkey anti-goat secondary (1:500, Invitrogen A-11055) or antibodies conjugated with Alexa Fluor 488 or donkey anti-rabbit IgG conjugated with Alexa Fluor 647 (1:500, Invitrogen A31573) in a dark chamber for 1 h at RT. Nuclei were stained by 4,6-diamidino-2-phenylindole (DAPI). Images were captured using Nikon Fluorescent microscope.
3
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).
4
Whole-Mount Imaging of Lung Vasculature
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Lungs were harvested at E12.5, E13.5, E15.5, E17.5 and E18.5 from both heterozygous (Cxcl12DsRed/+) and homozygous knockout (Cxcl12Dsred/DsRed) mice. Based on previously established protocols (Das et al., 2019 (link)), lungs were fixed in 2% paraformaldehyde for 4 h and then imaged for DsRed to evaluate the arterial endothelium or prepared for immunofluorescence-based whole-mount imaging. Lung lobes were incubated with primary antibody (rabbit anti-VWF, 1:100, F3520, Sigma-Aldrich; goat anti-EMCN, 1:200, AF4666, R&D Systems), in 5% donkey serum and 0.5% Triton X-100 in PBS (PBST) for 4 h at room temperature (RT) on a shaker, and overnight at 4°C on a shaker. The samples were washed in 0.5% PBST (four 30 min washes at RT), then overnight at 4°C on a shaker. The samples were then incubated in secondary antibody (donkey anti-mouse Alexa Fluor 488, A-21202 and anti-goat Alexa Fluor 647, A21447, Thermo Fisher Scientific, 1:250), in 5% donkey serum and 0.5% PBST for 1 h at RT on a shaker followed by overnight at 4°C. The lungs were washed in 0.5% PBST (four 30 min washes at RT), fixed in 2% paraformaldehyde for 2 h at RT and then washed with PBS (two 30 min washes at RT). Finally, the lungs were cleared using CUBIC (RIA reagent) overnight at 37°C, and then stored at 4°C until imaged using a Leica DMi8 Thunder system and/or Leica DMi8 confocal microscope.
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