Samples were fixed for 30 minutes with 4% paraformaldehyde (Electron Microscopy Sciences) diluted in phosphate-buffered saline (PBS, pH 7.2). Samples were washed 3x with fresh PBS and then blocked for 30 minutes with 10% goat serum (Invitrogen), with 0.2% triton X-100 (EMD Millipore) to permeabilize the cell membrane. RGCs were stained for βIII tubulin(mAB, 1:500, Covance) and Netrin-1 (pAB 1:500, R&D Systems) overnight at 4°C, washed 3x with PBS and labeled with Alexafluor 546 goat anti-mouse IgG (1:500, Invitrogen) and Alexafluor 488 goat anti-rat IgG (1:500, Invitrogen). Samples were imaged using a TCS SP5 Inverted Confocal Microscope (Leica) with mosaic images stitched using LAF software (Leica).
Laf software
Manufactured by Leica
LAF software is a computer program developed by Leica to manage and analyze data from laboratory equipment. It provides tools for data collection, processing, and visualization without making claims about its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Tcs sp5 inverted confocal microscope, by Leica (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rat igg, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Triton x 100, by Merck Group (1 mentions)
2 protocols using laf software
1
Immunostaining of Retinal Ganglion Cells
2
Wound Healing Assay in Cultured Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Cells were seeded in 35 mm culture dishes (density 2 × 105 cells per well). An incision was made after 24 hours in the central region of confluence in the culture dish. After an additional 48 hours, the dish was carefully washed to remove detached cells. Fresh medium was added. Cultures were observed at the time of incision and after 48 hours. Phase-contrast microscopy pictures were taken of 6 separate fields of the incised region. The distance between two broad edges of cells was measured and analyzed by Leica LAF software.
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