Epidermal club cell densities in fathead minnows: assessment of method and application to a case study

Abstract
Many fishes possess epidermal club cells that are the presumptive source of chemical alarm cues and also play a role in innate immune defense. Club cell density has been estimated in some studies but a standardized method for quantifying these cells is lacking.
Here, we assess the repeatability of estimating club cell density in fathead minnows (Pimephales promelas). Thin-sectioned histological samples of fathead minnows were stained and mounted on slides and then digitally scanned for scoring. We estimated epidermal area using the segment tool in ImageJ to simulate the traditional method of scoring microscope slides using an ocular micrometer, where epidermal area was estimated by the lengths of straight-line segments of epidermal thickness and length of the tissue sample. The second approach measured epidermal area using the freehand tool in ImageJ.
The R2 value for repeated estimates of club cell density (club cells/mm2) ranged from 0.959 and 0.969, depending on the method u..., Sample preparation
Adult wild-caught fathead minnows collected from local prairie pothole lakes near Fargo, North Dakota, USA were obtained from a local bait dealer. Laboratory-reared fathead minnows were obtained from the U.S. Environmental Protection Agency (EPA) in Duluth, MN, USA. Fish were sacrificed using a lethal dosage of methane tricainesulfonate (~500 mg/L; IACUC protocol #A18054) and a 3-4 mm section of skin was excised from the nape from eight wild-caught minnows and 11 lab-reared minnows. Samples were fixed in 10% formalin solution for 24 h then set in parafilm for 8 h. Samples were stained with periodic acid Schiff reagent and counterstained with hematoxylin (PAS-H) and subsequently thin-sectioned and mounted on slides.
Digitization and estimation of club cell density
Slides were digitally scanned using a MoticEasyScan slide scanner using Plan Apochromatic objective (20X/0.75) with image detail equivalent to 40X lens. Club cells appear as large, unstained, circular cells w..., , # Epidermal club cell densities in fathead minnows: assessment of method and application to a case study

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## Description of the Data and file structure

The excel file has two tabs. The first tab contains from the first counts, the second tab contains data from the second counts.

Explanation of column headings:
Slide = label on the slides we scored;
Wild1Lab2 = code for source population, 1 = wild-caught fathed minnows from a bait store, 2 = lab-reared fathead minnows from the EPA labs in Duluth, MN, USA;
pixels_100um_scale = using imageJ, the number of pixels in the image that spanned the scale marker for 100um;
pixels/mm = conversion factor for measurements in pixels to mm and mm2
CC = number of epidermal club cells
Area_by_FHTool_pixels = raw data in pixels for area of enclosed epidermal tissue using the freehand tool in imageJ;
AreaFHTmm = epidermal area based ondata from the freehand tool in mm2
Thickness = epidermal thickness in pixels
Thicknessmm = epidermal thickness in mm...