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C-fos like immunoreactive (c-fos LI) nuclei in the trigeminal brain stem complex were identified under bright field microscopy and counted by an observer naïve to the treatment as previously described [31, 34].
Apoptotic cells were labeled using a TUNEL assay kit according to the manufacturer's instructions (Roche, Nutley, NJ), imaged using an epifluorescence microscopy, and counted.
The 24-well microtiter plates were rinsed gently at least six times with sterile phosphate buffer solution (pH 7.5) and strained by 0.1% crystal violet for 20 min. Adherent bacterial cells were observed by phase contrast microscopy and counted.
All characteristic colonies were checked by microscopy and counted (Arla Foods amba).
TUNEL-positive cells were visualized by immunofluorescent microscopy and counted using a 20× objective.
For quantitative determination, clusters in 50 randomlyselected myotubes in two separate experiments were viewed by fluorescence microscopy and counted.
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Coded slides were double labeled for Ki67 and DRAQ5 and processed for confocal microscopy and counting as previously described (He et al. 2000).
Parasite counts were determined by microscopy and counting infected erythrocytes per 1,000 erythrocytes in thin blood films, or calculated from the parasite count per 200 leukocytes in thick blood films.
Slides were triple labeled with an anti-BrdU antibody, the nuclear stain DRAQ5, and antibodies for rods, BCs, or MGCs and then processed for confocal microscopy and counting as previously described (He et al. 2003; Johnson et al. 2007).
Immunostaining was quantitatively evaluated by two different observers in a blinded fashion (DP, RB) using light microscopy and counting the positive cells across three high power fields (HPF at 40x magnification), each containing about one hundred cells.
Five days after treatment, cells were photographed (phase-contract microscopy), harvested, and counted using a hemocytometer.
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