Quorum k850 critical point dryer

For SEM, fresh sperm isolated from cauda epididymis of mice were washed three times using PBS and then fixed on coverslips with 4% Paraformaldehyde for 2 h. Subsequently, the coverslips carrying spermatozoa were washed with PBS and then dehydrated with gradient ethanol of 50, 60, 70, 80, 95, and 100%. Then, the sperm were dried with Quorum K850 Critical Point Dryer (East Sussex, UK) and coated with gold particles using an ion sputter coater (Rotary Pumped Quorum Technologies, Q150RS). The spermatozoa were finally observed under SEM (Hitachi, S-3400N).
For TEM, testis tissues were isolated from mice and then fixed with 2.5% glutaraldehyde for more than 24 h. Subsequently, the samples were further fixed with 1% osmic acid for 120 min. After washing with PBS twice, the samples were dehydrated with gradient acetone of 50, 70, 90, and 100% at 4 °C and then embedded in embedding agents containing 6% butylene phthalate, 1% phenol, 44% dodecyl succinic anhydride, and 56% epoxy resin. Next, ultrathin sections (70–90 nm) were obtained and double-stained with lead citrate and uranyl acetate. Finally, the images were taken under transmission electron microscopy (Philips, TECNAI G2 F20) for further analysis.

Fresh leaves from each developmental stage and stems (≈10 each) were rinsed in a 1% sodium hypochlorite solution to clean surfaces, and the abaxial surface of some leaves was stripped with cellophane tape to view the underlying trichomes. Thereafter, leaf and stems were cut into smaller sections (≈3 mm × 4 mm) and fixed in 2.5% glutaraldehyde for 24 h at 4 °C before being subjected to three phosphate buffer (0.1 M with a 7.2 pH) washes (5 min each). This was followed by post-fixation in 0.5% osmium tetroxide for 4 h at room temperature. Thereafter, the material underwent three phosphate buffer rinses (5 min each) and was then dehydrated in a graded series of ethanol, 30%, 50%, 70% (each twice for 5 min), and 100% (twice for 10 min). Following dehydration, samples were dried with a Quorum K850 Critical Point Dryer. Chemically fixed leaf and stem fragments were then mounted onto aluminum stubs which were secured with carbon conductive tape and sputter-coated with gold in a Quorum Q150 RES gold Sputter Coater. Samples were viewed using a Zeiss Leo 1450 SEM (Oberkochen, Germany) at a working distance of 18 mm. Images were captured using the SmartSEM imaging software.