Cells were grown for 24-48 hours on sterilized coverslips under standard growth conditions and fixed in 4% vol/vol para-formaldehyde (PFA) in 0.25 M HEPES for 15 minutes, followed by permeabilization in 0.2% vol/vol Triton X-100 in PBS for 10 minutes. Following blocking with 10% vol/vol fetal calf serum in PBS, von Willebrand’s factor (VWF) was detected using mouse anti-VWF 1:100 (Clone F8/86, MA5-14029, Invitrogen) and goat anti-mouse Alexa Fluor 488 1:500 (A32723, Thermo Fisher Scientific). DNA was stained with 1 μg ml-1 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI) in PBS and after washing coverslips were mounted in Vectashield (Vector Laboratories). Widefield fluorescence imaging was performed on a DeltaVision Elite system (Applied Precision) using a UPLFLN 40× 1.30NA oil immersion objective (Olympus), a CoolSnap HQ2 CCD camera (Photometrics), DAPI (excitation 390/18; emission 435/40) and FITC (excitation 475/28; emission 525/45) filters. 12-bit image stacks were acquired with a z-step of 200 nm giving a voxel size of 161.3 nm × 161.3 nm × 200 nm. All images were acquired using the same exposure settings. Using Fiji103 (link), 3D images were flattened by maximum intensity projection and displayed at the same minimum/maximum intensity settings. Images were cropped for publication in Adobe Photoshop (v22.4.1).
Uplfln 40 1.30na oil immersion objective
Manufactured by Olympus
The UPLFLN 40× 1.30NA oil immersion objective is a high-numerical aperture lens designed for use in optical microscopy. It provides a magnification of 40× and a numerical aperture of 1.30, which enables high-resolution imaging. The objective is optimized for use with oil immersion to enhance the optical performance.
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2 protocols using uplfln 40 1.30na oil immersion objective
1
Immunofluorescence Imaging of von Willebrand Factor
2
Immunofluorescence Imaging of von Willebrand Factor
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Cells were grown for 24-48 hours on sterilized coverslips under standard growth conditions and fixed in 4% vol/vol para-formaldehyde (PFA) in 0.25 M HEPES for 15 minutes, followed by permeabilization in 0.2% vol/vol Triton X-100 in PBS for 10 minutes. Following blocking with 10% vol/vol fetal calf serum in PBS, von Willebrand’s factor (VWF) was detected using mouse anti-VWF 1:100 (Clone F8/86, MA5-14029, Invitrogen) and goat anti-mouse Alexa Fluor 488 1:500 (A32723, Thermo Fisher Scientific). DNA was stained with 1 μg ml-1 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI) in PBS and after washing coverslips were mounted in Vectashield (Vector Laboratories). Widefield fluorescence imaging was performed on a DeltaVision Elite system (Applied Precision) using a UPLFLN 40× 1.30NA oil immersion objective (Olympus), a CoolSnap HQ2 CCD camera (Photometrics), DAPI (excitation 390/18; emission 435/40) and FITC (excitation 475/28; emission 525/45) filters. 12-bit image stacks were acquired with a z-step of 200 nm giving a voxel size of 161.3 nm × 161.3 nm × 200 nm. All images were acquired using the same exposure settings. Using Fiji103 (link), 3D images were flattened by maximum intensity projection and displayed at the same minimum/maximum intensity settings. Images were cropped for publication in Adobe Photoshop (v22.4.1).
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