Diff quik stain

After measurement of AHR, the mice were euthanized and BALF was collected from the right lung after tying off the left lung at the mainstem bronchus. The right lung was lavaged three times with 0.4 mL PBS per wash. In some studies, BALF was collected from both lungs by lavaging four times with 1 mL PBS per wash. Total BALF cell numbers were counted with a hemacytometer, the fluid was centrifuged at 200 × g for 10 min at 4°C, and a Cytospin slide of resuspended cells was prepared. Eosinophils were quantified via light microscopy using Diff-Quik stain (Dade Behring) and morphological criteria. Eosinophil percent was expressed as percent of total BALF cells and as percent relative to the vehicle control in each study.

All men produced their semen sample in a sterile labeled jar. The semen sample was produced via masturbation on the day of oocyte aspiration after 2–5 days of abstinence, and the collected semen sample was left to liquefy at 37 °C for 30 min before analysis. Each sample was split into two aliquots. One of these was subjected to analysis for seminal characteristics. Standard semen parameters were assessed according to WHO 2010 standards; to summarize, the sperm number was determined using an upgraded Neubauer chamber after proper dilution. Motility was determined using a Leica microscope DM300 scoring at least 100 spermatozoa/slide, and morphology was determined using Diff-Quik stain (Dade Behring Inc., Newark, NJ, USA). The other aliquot used was for sperm preparation. The final acquired fraction was tested for sperm count and motility, and then it was maintained at 37 °C in the same medium for 15 min until used for inseminating oocytes through ICSI or IVF. Sperm HOS, ROS, sperm DNA fragmentation (SCD) and chromatin maturity (CMA3) were evaluated in selected spermatozoa remaining after oocyte insemination.

CTVT and MCTVT samples were surgically excised from tumour-bearing beagles, and XCTVT-bearing mice were euthanized with isoflurane and cervical dislocation to obtain their tumours. Single-cell suspensions were prepared from the tumour samples following the procedures described above. Tumour cell smears were stained with Diff-Quik stain (Dade Behring, USA) for subsequent cytological examination [43 (link)]. For histopathological analysis, tumours were fixed with 10% buffered formalin for 24 h and embedded in paraffin. Deparaffinized tissue sections (4 μm thick) were stained with hematoxylin and eosin and the mitotic index was determined by enumerating mitotic figures in 10 consecutive fields at 40x objective magnification in each sample. Slides were independently and separately scored by two board-certified veterinary pathologists from the NTU veterinary hospital who were blinded to the experiment.

Cell motility was determined using 8 micron PET membrane transwell culture chambers (BD Biosciences, San Jose, CA). Cells were serum-starved overnight. RPMI media/10% serum (500 μl) was added to lower wells and cells were seeded in 350 μl serum-free RPMI media in the upper wells. Recombinant CTGF (5 μg/ml), FG-3019 (100 μg/ml) and/or IgG (100 μg/ml) were added to the top and bottom wells, and the chambers were incubated at 37°C for 6 hours. The non-motile cells were removed from the upper surface of the membrane with a cotton-tipped swab. The membranes were then fixed and stained using Diff-Quik stain (Dade Behring, Deerfield, IL). Three independent experiments were performed with triplicate samples. The number of migrating cells was calculated by counting the total number of cells in 5 fields at 20X magnification.

Neonatal BALF and primary AM were isolated from the pups using methods previously described by our laboratory (Gauthier et al. 2010). Briefly, the pups were anesthetized with sodium pentobarbital intraperitoneally. Using a dissecting microscope, the pup trachea was identified, cannulated with a 27G catheter, and the lungs serially lavaged with sterile phosphate‐buffered saline. The lavage from each pup of the same genotype was pooled (WT vs. KO) and centrifuged (402g for 8 min). The cell‐free BALF was saved for further analysis. The retrieved cells were evaluated for cell type using DiffQuik stain (Dade Behring, Newark, DE).