Aniline blue

For spray inoculation, conidial suspension (10 ml) containing Tween 20 (250 ppm) and conidia harvested from 12-day-old cultures on OMA plate (1–5×10⁵ conidia/ml) was sprayed onto four-weeks old susceptible rice seedlings (Oryza sativa cv. Nakdongbyeo). Inoculated plants were placed in a dew chamber at 25°C for 24 hours in the dark, and then transferred back to the growth chamber with a photoperiod of 16 hours using fluorescent lights [74] . Disease severity was assessed at seven days after inoculation. The %DLA was recorded to permit more accurate evaluation of the virulence of the mutants. Photographs of diseased rice leaves including eight centimeter long leaf blades were taken. The number of pixels under lesion areas and healthy areas of diseased leaves was calculated by Axiovision image analyzer with the photographs. For microscopic observation of penetration and infectious growth on rice tissue, excised rice leaf sheath of Nakdongbyeo were prepared as previously described [30] (link),[42] and inoculated by conidia suspension (1×10⁴ conidia/ml) on the adaxial surface. After 24, 48 and 96 hours incubation in a moistened box, the sheaths were trimmed to remove chlorophyll enriched plant parts. Remaining epidermal layer of mid vein (three to four cell layers thick) were utilized for microscopic observations. Inoculation on onion epidermis was performed as previously described [75] (link). Fixation and aniline blue staining of rice sheath and onion epidermis were performed as previously described [75] (link). Samples were incubated in lactophenol at room temperature for 1hour and directly mounted with 70% glycerin or transferred into 0.01% aniline blue for 1hour and destained with lactophenol. For 3, 3′-diaminobenzidine (DAB, Sigma, D-8001) staining, samples were incubated in 1mg/ml DAB solution (pH 3.8) at room temperature for 8 hours and destained with clearing solution (ethanol∶acetic acid = 94∶4, v/v) for 1 hour. For observation and scoring penetration rate and IH development, conidia suspension were dropped on onion epidermis and incubated for 72 hours in moistened culture plate. Samples were fixed and stained as rice sheath described above. Extensive IH from single appressoria with no (or scatterd) callose were scored as normal IH, relative short and attenuated IH with accumulated callose were scored as retarded IH, appressorium developing very short IH or penetration peg with strong callose were scored as blocked IH, and appressorium without IH and callose deposition were scored no penetration.

AnimalsIn this experimental study, totally 20 adult male Syrian mice (10 weeks old, 35g) were divided into 2 groups of A and B. The mice of group A considered as controls with no medication, whereas group B (diabetic mice) received diabetogenic agent, Streptozotocin (STZ) and basal diet. Both groups were housed in a controlled environment with a temperature range of 25±3^oC and mean relative humidity of 50±5%. They were fed mice chow and had access to water ad libitum. This experimental project was approved by ethical committee of Shahid Sadoughi University of Medical Sciences.
Induction of diabetesIn experimental group, the diabetes was induced via a single intra-peritoneal (i.p.) injection of buffered solution (0.1 mol/l of citrate, pH 4.5) of STZ at a dosage of 200 mg/kg body weight. At this dose, STZ induces considerable hyperglycemia (blood glucose > 250 mg/dl) in mice that was measured at 72 hours post-injection (4 ). After that, the blood glucose was measured periodically with the mean value 275 mg/dl in diabetic mice.
Epididymal sperm preparationAfter 35 days (one duration of spermatogenesis in mice is about 32 days), a small part of the cauda epididymis of each mouse was dissected and located in 1 mL of pre-warmed Hams F10 medium (37^oC, 5% CO₂). Gentle tearing of the tissue was done to make spermatozoa swim out into the culture medium. The dishes were placed in the incubator for 15 min.
Sperm analysisThe sperm motility, normal morphology, viability and sperm count were evaluated for at least 200 spermatozoa of each animal. Sperm movement analysis was done by Makler Chamber and light microscopy (Olympus Co., Tokyo, Japan). Motility was expressed as the percentages of progressive motility including Rapid (Grade a) and Slow (Grade b) spermatozoa, non-progressive (Grade c) and immotile (Grade d) spermatozoa. The morphologically normal spermatozoa and the percentage of viable sprm cells were assessed by Papanicula staining and Eosin test respectively (10 ). The light microscope was set at ×40 eyepiece magnification. All analyses were performed by one experienced technician blinded to the study. Double checking of results was also done for each specimen.
Sperm chromatin/DNA evaluationDNA integrity and chromatin condensation assessments were assessed by standard cytochemical techniques including Acridine Orange Test (AOT), Aniline Blue (AB), Toluidine Blue (TB) and Chromomycin A3 (CMA3). All dyes and chemicals were purchased from Sigma Aldrich Company (St Louis,MO, USA). The efficacy of dyes was tested with and without acid denaturation of normal samples and they were considered as positive and negative controls, respectively (12 (link)).
a. Aniline Blue (AB) stainingAniline blue selectively stains lysine-rich histones and is able to show those sperm chromatin condensation anomalies that are related to residual histones. To do this staining, air-dried smears from washed semen samples were fixed in 3% buffered glutaraldehyde in 0.2 M phosphate buffer (pH 7.2) for 30min at room temperature. Each smear was stained with 5% aqueous AB stain in 4% acetic acid (pH=3.5) for 7 min. In light microscopic evaluation, 200 spermatozoa were counted in different areas of each slide using ×100 eyepiece magnification (8 ).
b. Toluidine Blue (TB) stainingToluidine blue is a metachromatic dye which shows both the quality and the quantity of sperm nuclear chromatin condensation/ DNA fragmentation via binding to phosphate groups of DNA strands (7 ). Briefly, air-dried sperm smears were fixed in fresh 96% ethanol and acetone (1:1) at 4^oC for 30 min and then incubated in 0.1 NHCl at 4^oC for 5 min. After that, the slides were washed 3 times with distilled water for 2 min and finally stained with 0.05% TB in 50% citrate phosphate for 10 min at room temperature. In each sample, at least 200 spermatozoa were counted under light microscopy with ×100 eyepiece magnification (13 (link)).
c. Acridine Orange Test (AOT)Acridine orange is a metachromatic fluorescence probe for demonstration of degree of sperm nuclear DNA susceptibility to in-situ acid-induced denaturation by distinction between native double-stranded DNA (green fluorescent) and denatured single-stranded DNA (red fluorescent). Briefly, the air-dried smears were fixed in Carnoy’s solution (methanol/glacial acetic acid, 3:1) at 4^oC for at least 2 hrs. Each sample was stained by freshly prepared AO (0.19 mg/ml in McIlvain phosphate-citrate buffer (pH=4) for 10 min. Smears were assessed on the same day using fluorescent microscope (Zeiss Co., Jena, Germany) with a 460-nm filter (8 ).
d. Chromomycin A3 stainingCMA3 is a fluorochrome specific for guanosine cytosine-rich sequences and it is used for estimate of the degree of sperm chromatin protamination (11 ). For this purpose, the smears were dried first and then fixed in Carnoy’s solution at 4^oC for 10 min. The slide was treated with 150µl of CMA3 (0.25mg/ml) in McIlvain buffer for 20min. After staining in darkroom, the slides were washed in buffer and mounted with buffered glycerol. In each sample, at least 200 spermatozoa were counted under fluorescent microscope with a 460-nm filter and ×100 eyepiece magnifications (14 (link)).
Statistical analysisStatistical analysis was performed by SPSS software version 18 for Windows (SPSS Inc., Chicago, IL, USA). Student’s t-test was applied to evaluate the data and the term ‘statistically significant’ was used to signify a two-sided p<0.001 for sperm parameters and cytochemical tests.

The visualization of growing PTs with aniline blue staining utilizes the ability of this fluorochrome to bind to the callose contained in the pollen grain wall and PTs.
The pollinated pistils fixed in acetic alcohol (90% ethanol and acetic acid, 3 : 1) were used in the experiments. Pistils were macerated in 20% KOH alcohol solution for 20–40 min, washed twice with distilled water, and stained with 0.01% aniline blue solution for 30–40 min. The stained pistils were placed into a drop of glycerin mixed with water (1 : 1) on a glass slide, covered with a cover glass, gently squashed, and examined using a Zeiss Axioplan (Carl Zeiss, Germany) fluorescence microscope with 365 nm excitation filter and 420 nm emission filter. At least 200 PTs were examined in each variant of the experiment.

Staining for callose was carried out on thick sections (1 μm) of material embedded in LR White resin. Samples were placed in 1% aniline blue (a fluorescent dye that localizes callose, Smith and McCully, 1978 (link)) in 0.067 M Na₂HPO₄ buffer (pH 8.5) in a dark, humid chamber at 4°C for 3 nights, followed by 3 rinses in buffer. Controls were placed in buffer without stain. All stained material was viewed with a Leica DM500B compound microscope (excitation filter ultraviolet fluorescence between 360-400 nm). Images were collected digitally using a Q-Imaging Retiga 2000R digital camera.​ Control sections lack fluorescence entirely at the apex or show general cell wall autofluorescence in mature stem regions and therefore are not included in the illustrations.