To determine the Paneth cell-specific expression of DEFA6, immunohistochemistry was performed by the Benchmark Ultra system (Ventana) and the ultraView Universal DAB Detection Kit (Ventana) and even counterstaining
with hematoxylin-eosin was carried out.
DAPI (4',6-diamidino-2-phenylindole) was used for nuclear counterstaining
For tagging, chromogen diaminobenzidine (DakoCytomation, Carpinteria, CA, USA) was used for 10 min, and Mayer's Hematoxylin was used for the counterstaining
. Reactions were performed with negative and positive controls.
Immunostaining of CD45 and counterstaining
with hematoxylin; scale bar =100 [micro]m.
The appearance of DAPI enhanced regions subsequent to FISH procedure and DAPI counterstaining
(FISH DAPI+ bands), that in fact mimic the C-banding pattern, is described in Moscone et al.
Slides were dipped in a solution of haematoxylin that is diluted 1:1 in distilled water and stained for one minute to produce a very light nuclear counterstaining
. Washed for 1 min.
Biotin-conjugated goat anti-rat IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was used as a secondary antibody, followed by incubation with a streptavidin-biotin complex (Vector Laboratories, Burlingame, CA, USA) and subsequent development with 0.4% 3-amino-9-ethylcarbazole for 5 minutes and counterstaining
with Gill's Haematoxylin.
The percentages of replicated [beta]-cells were determined by immunofluorescent assays using anti-insulin and anti-BrdU or anti-Ki67, followed by counterstaining
We then allowed the color to develop with DAB for 30 min, by adding the peroxidase-labeled streptavidin dropwise, incubation at 37[degrees]C for 20 min, followed by mounting after counterstaining
For nuclear counterstaining
, the cells were treated for 5 min with 4',6-diamidino-2-phenylindole (DAPI) (Beyotime Bio., China) solution.
of stereocilia was performed using phalloidin labeled with tetramethyl rhodamine isothiocyanate (TRITC-phalloidin, 1:200; Sigma, St.
To detect the distribution of iron particles in the tumor, either within tumor vessels or taken up by macrophages, both anti-CD31 and anti-CD68 (Abcam) immunohistochemistry with iron particles counterstaining
were performed on one tumor model randomly selected from each treated subgroup.
was performed by DAPI staining and a total of three hundred metaphase cells were microscopically analyzed for each probe set employed.
The biopsy material was stained with Silver Methenamine with H&E counterstaining
. Gram-positive hyphae/pseudohyphae can be appreciated.
Slides were rinsed twice in PBS (10 min each), and nuclei were then distinguished by counterstaining
with Hoechst 33258 stain (Sigma-Aldrich, Darmstadt, Germany).