Poly l lysine coated slide

Frozen sections of spleen (thickness, 6 mm) were obtained using a cryostat (temperature, −22°C) (Thermo Fisher Scientific, USA). The sections were fixed in acetone for 5 min using poly-L lysine-coated slides (Sigma-Aldrich). Inside a humidifying chamber, the tissues were stored in PBS/10% horse serum ([HS] to prevent non-specific staining) (Thermo Fisher Scientific) overnight at RT. After incubation and three washes with 1×PBS, 80 μL of a primary antibody/diluent was added and the sections were incubated for 30 min at RT. The sections were washed again and 80 μL of biotinylated horse anti-mouse immunoglobulin (Ig) G was added as the secondary antibody (Thermo Fisher Scientific).
The sections were incubated for 30 min at RT followed by 5 washes with PBS. Then, 80 μL of avidin-biotin complex reagent was immediately added and the sections were incubated for 30 min at RT. The sections were washed (5 times) and 100 μL of “charged” DAB (3, 3’-diaminobenzidine) (Abcam, USA) was added to each slide for color development. After a final course of washing, methyl green was added to the sections, which were then incubated for 1 h. The slides were dipped in tap water and passed through a series of dehydrating baths of ethanol as follows: 70%, 95%, and 100% for 30 s, 100% ethanol-100% Americlear (50:50 mix) for 15 s, and 100% Americlear for 1 min [21 (link),22 ].

The fixed testes were dehydrated through a series-graded concentrations of ethanol, clarified in xylene, embedded in paraffin, sectioned at 4 μm, and placed on poly-l-lysine-coated slides (Sigma) for hematoxylin and eosin staining. Sections were examined by light microscopy. The criteria used for evaluation of histopathological changes of testes were followed from the principles described by Hess et al. (1988 (link)). An average number of 250 seminiferous tubules per animal (n = 4) were examined as reported by Thompson et al. (2009 (link)) and testicular degeneration was qualitatively assessed and scored into 4 scores as follows: normal (1), mild (2), moderate (3), and severe (4).

For P75 and VERSICAN detection, hindguts were fixed in 4% formaldehyde in PBS for 1 h and infiltrated with 15% sucrose/PBS overnight at 4 °C. The medium was changed to 7.5% gelatin containing 15% sucrose at 37 °C for 1 h and the tissues were rapidly frozen at −50 °C in methylbutane (Sigma-Aldrich, Budapest, Hungary). Frozen sections were cut at 12 μm thickness, collected on poly-l-lysine-coated slides (Sigma-Aldrich, Budapest, Hungary) and stained by immunocytochemistry as previously described [60 (link)]. Briefly, after rehydration, sections were incubated with primary antibodies for 1 h. Primary antibodies used were anti-p75 ^NTR (1:2000, kind gift of Dr. Louis Reichardt [61 (link)]) and anti-Versican (1:500, kind gift of Dr. Maria T. Dours-Zimmerman [62 (link)]). Sections were incubated with biotinylated goat anti-rabbit IgG (1:200, Vector Laboratories BP-9100-50, Burlingame, CA, USA) for 45 min and avidin-biotinylated peroxidase complex (Vectastain Elite ABC kit, Vector Laboratories PK-6105, Burlingame, CA, USA) for 30 min at room temperature. Endogenous peroxidase activity was quenched by incubation for 10 min with 3% hydrogen peroxide (Sigma-Aldrich, Budapest, Hungary) in PBS. Binding sites of the primary antibodies were visualized by 4-chloro-1-naphtol (Sigma-Aldrich, Budapest, Hungary).

Plant materials were photographed with a Nikon E995 digital camera (Nikon, Japan) mounted on a Motic K400 dissecting microscope (Preiser Scientific, Louisville, KY, USA). Fresh microspores were stained with 1% acetocarmine and 2% iodine-potassium iodide (2% I₂-KI). Anthers at different developmental stages were fixed in FAA and embedded in paraffin wax. Transverse sections of 6 μm were placed onto poly-L-lysine-coated slides (Sigma-Aldrich), and stained with safranin O/fast green and Ehrlich’s hematoxylin. For 4’, 6-diamidino-2-phenylindole (DAPI) staining of the nuclei, transverse sections and microspores were washed, embedded, and stained as described previously [37 ]. Samples were photographed using a DS-U2 high resolution camera mounted on a Nikon ECLIPSE E600 microscope along with NIS-Elements software (all from Nikon).
For transmission electron microscopy observation, anthers were fixed, embedded, and stained according to Cheng et al. [7 ] and examined with a JEM-1230 transmission electron microscope (JEOL). For scanning electron microscopy, anthers and microspores were collected and processed essentially as described by Zhang et al. [38 (link)], and observed with a JSM-6360LV scanning electron microscope (JEOL).