All the slides were examined at ×600 magnification using a Nikon Optiphot 2 fluorescence microscope, equipped with separate filters for DAPI and fluorescein isothiocyanate (FITC). Manual scoring was timed for three unirradiated and three 1 Gy/1 h incubated samples on slides produced by both processing methods. A total of 50 lymphocytes were scored per sample and the time taken to do this was recorded every ten cells. To ensure the lyse/fix processing itself did not affect foci levels, samples irradiated at 0.5 Gy/repair time 30 min and prepared using both protocols were used to assess the number of foci per cell seen in a panel of 17 donors. 50 lymphocytes were scored in each of the reference samples and the foci numbers were used to adjust the calibration coefficients and the associated standard errors (Rothkamm et al., 2013b (link)) of the laboratory’s calibration curve (Horn, Barnard & Rothkamm, 2011 (link)). Blood dose estimates for the unknown samples were produced by scoring up to 50 lymphocytes or 200 foci per sample.
Optiphot 2 fluorescence microscope
Manufactured by Nikon
The Optiphot 2 fluorescence microscope is a laboratory equipment designed for fluorescence microscopy. It provides a core function of enabling the observation and analysis of samples using fluorescence imaging techniques.
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4 protocols using optiphot 2 fluorescence microscope
1
Quantifying DNA Damage Foci in Lymphocytes
2
Immunohistochemical Staining of Cone-Rich Retinal Sections
3
Amyloid Plaque Identification via Thioflavin S
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Slides from a subset of subjects were labeled with thioflavine S to verify whether plaques contained amyloidogenic fibrils. Sections processed for thioflavine S were defatted in 1:1 chloroform and 100% ethanol for 2 h, rehydrated as above, and stained with 0.5% aqueous thioflavine S (Sigma-Aldrich) for 20 min at RT, followed by differentiation in 80% ethanol. After several dH2O rinses, slides were coverslipped with an aqueous mounting medium (Gel-Mount; Biomeda, Foster City, CA, USA). Thioflaving S was visualize with the aid of a Nikon Optiphot-2 fluorescence microscope.
4
Micronucleus Induction Assay with PARP, ATR, and CHK1 Inhibitors
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Cells (2 × 106) were seeded onto 100 mm Petri dishes and cultured for 24 h before treatment with olaparib, ATRi, CHK1i, and a combination of PARPi with ATRi or PARPi with CHK1i, each at a concentration of 4 µM. After 48 h of exposure, cells were washed twice with PBS, collected and incubated in a chilled hypotonic solution of KCl (75 mM) for 30 min. Samples were then centrifuged at 200× g for 5 min at 25 °C and fixed with Carnoy’s fixative (3:1 ratio of methanol:glacial acetic acid) for 5 min. After centrifugation at 200× g for 5 min, cells were resuspended in fresh Carnoy’s fixative. This step was repeated twice. Finally, cells were placed on microscopic slides, fixed with methanol for 15 min, air dried and stained with acridine orange for 2 min (10 μg/mL in PBS). Micronuclei (MN) were examined under an Optiphot-2 fluorescence microscope (Nikon) equipped with a B-2A filter (blue light; λ = 495 nm). The MN images presented were visualized by fluorescence microscopy (Olympus IX70, Tokyo, Japan; magnification 400×).
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