Leitz aristoplan microscope

Retina cross-sections were mounted and stained with toluidine blue. Images of whole retina cross-sections were obtained using Leica leitz Aristoplan microscope equipped with DFC 480 camera (Leica) and LAS 3.7 version software. Thickness of the different nuclear and synaptic layers were determined using Visilog 6.4 version software (Noesis).

This test verifies the maturity of the sperm chromatin, since AB shows binding affinity with lysine residues, an amino acid in which histones are rich. It is well known that, during the maturation process, spermatozoa replace most histones with protamines, basic proteins rich in arginine accomplishing chromatin compaction. Therefore, the blue staining of sperm after AB treatment indicates the persistence of histones and the immaturity of spermatozoa.
Two hundred microliters of seminal fluid were washed in phosphate buffer saline (PBS), centrifuged at 400× g for 15 min, and resuspended in PBS; the sample, appropriately diluted, was smeared on slides and air-dried. The slides were fixed with 3% glutaraldehyde in PBS, pH 7.2, for 30 min in a humid chamber at room temperature. Then, the slides were treated for 5 min with AB solution (5% aniline powder and 4% acetic acid, with solution made up to volume with distilled water; pH 3.5). After washing in distilled water, the slides were observed under a Leitz Aristoplan microscope (Leica, Wetzlar, Germany). Approximately 300 spermatozoa per sample were examined.

Free-floating sections were incubated (overnight, room temperature) with rabbit anti-Ki67 (1:1000, Leica, Cedarlane, Burlington, ON, Canada) or goat anti-doublecortin (DCX, 1:1000, Santa Cruz), then incubated in biotinylated species-specific secondary antibodies and the Avidin-Biotin complex (ABC kit, Vector laboratory). The immunoreactive material was detected in 3′-diaminobenzidine (DAB, brown precipitate). Immunoflurorescence staining with rabbit anti-calbindin (1:10000, Swant, Switzerland), anti-β-catenin (1:100, Santa Cruz), anti-Prox1 (1:3000, Millipore, Temecula, CA), or goat anti-DKK-1 (1:60, R&D system, Minneapolis) was detected with species-specific cyanin-3 (Cy3) or Alexa 594 (red) conjugated secondary antibody (Jackson Labs, West Grove, PA, USA). Sections (minimum 2-3/mouse) were observed under light microscopy or epifluorescence on a Leitz Aristoplan microscope (Leica, Montréal, QC, Canada), or confocal microscopy (LSM 510 or 710, Zeiss), digital pictures were taken and used for analysis. Staining specificity was confirmed by omitting primary antibodies.