Goat anti hamster igg alexafluor 568

For general histological analysis of the heart and lungs, E18.5 embryos were dissected while submerged under ice-cold PBS to prevent breathing of air and preserve developmental anatomy, and the thoracic cavity was exposed for overnight fixation with Bouin's solution, followed by paraffin embedding, serial microtome sectioning with a thickness of 5 µm, and staining with hematoxylin and eosin (H&E). For immunofluorescence experiments, lungs dissected as described above were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) overnight at 4°C, transferred to 30% sucrose for cryoprotection, embedded in Tissue-Tek optimal cutting temperature compound (Sakura Finetek), and sectioned at a thickness of 25 µm using a cryostat microtome (Leica). Tissue sections were stained overnight with the primary antibodies hamster anti-Podoplanin (DSHB 8.1.1; 1:800 dilution), rabbit anti-pro-Surfactant C (Millipore AB3786; 1:1500 dilution) and for 1 h at room temperature with the following fluorescent secondary antibodies: goat anti-hamster IgG AlexaFluor 568 (Invitrogen A-21112; 1:500 dilution), goat anti-rabbit IgG AlexaFluor 647 (Invitrogen A-21245; 1:500 dilution), and NucBlue Hoechst 33342 (Invitrogen; two drops/mL). Fluorescence microscopy was performed on a Nikon Ti widefield microscope.

For general histological analysis of the heart and lungs, E18.5 embryos were dissected while submerged under ice-cold PBS to prevent breathing of air and preserve developmental anatomy, and the thoracic cavity was exposed for overnight fixation with Bouin's solution, followed by paraffin embedding, serial microtome sectioning with a thickness of 5 µm, and staining with hematoxylin and eosin (H&E). For immunofluorescence experiments, lungs dissected as described above were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) overnight at 4°C, transferred to 30% sucrose for cryoprotection, embedded in Tissue-Tek optimal cutting temperature compound (Sakura Finetek), and sectioned at a thickness of 25 µm using a cryostat microtome (Leica). Tissue sections were stained overnight with the primary antibodies hamster anti-Podoplanin (DSHB 8.1.1; 1:800 dilution), rabbit anti-pro-Surfactant C (Millipore AB3786; 1:1500 dilution) and for 1 h at room temperature with the following fluorescent secondary antibodies: goat anti-hamster IgG AlexaFluor 568 (Invitrogen A-21112; 1:500 dilution), goat anti-rabbit IgG AlexaFluor 647 (Invitrogen A-21245; 1:500 dilution), and NucBlue Hoechst 33342 (Invitrogen; two drops/mL). Fluorescence microscopy was performed on a Nikon Ti widefield microscope.