Rm2235 rotary microtome

The other portions of corneas were prepared for microscopical examination according to Moreau et al. (1997[16 (link)]) by fixing immediately in 10 % neutral buffered formalin, followed by dehydration in upgraded concentrations of alcohol and immersion in xylene until clearance. Corneal tissues were then embedded in paraffin wax and the prepared blocks were cut into 4 mm thick sections using Leica™ RM2235 rotary microtome (Germany). Sections were stained with hematoxylin and eosin and observed under brightfield microscopy for tissue abnormalities.

The testes and ovaries were dissected and fixed in 4% paraformaldehyde (PFA) in PBS at 4 °C for 8 h. Tissues were dehydrated by treatment with an ethanol series (50%, 75%, 95%, 100%), cleared twice in xylene, and embedded in paraffin. Tissue sections (5 μm) were prepared with a Leica RM2235 rotary microtome. Sections were stained with hematoxylin and eosin according to the standard protocol (BBI, E607318). TUNEL assay was carried out with the TUNEL ApoGreen Detection Kit (Yeasen, 40307ES20).

The following instruments were used in this study: a RM2235 rotary microtome (Leica, Wetzlar, Germany); TEC-2500 histopathology dryer (Changzhou Hao Silin Instrument Equipment Co., Ltd., Nanjing, China); BX43 microscope (Olympus, Tokyo, Japan); PYX-DHS500BS-II water jacket constant-temperature incubator (Shanghai Yuejin Medical Instrument Co., Ltd.); BCD-211KD3 refrigerator (TCL, Huizhou, China); C21-SDHC15K induction heater (Zhejiang Shaoxing Supor Life Electric Co., Ltd., Hangzhou, China); and 101-3 electric heating constant-temperature dryer (Shanghai Jinping Instrument Co., Ltd., Shanghai, China).

Flies were aged and then fixed in Carnoy’s fixation solution (30% chloroform; 10% acetic acid; 60% absolute ethanol) for 3.5 h and flies were then placed in 95% ethanol for 30 min twice and then they were placed in 100% ethanol for 45 min. Flies were kept in methyl benzoate overnight. On the next day, flies were kept in solution of methyl benzoate and paraffin both in equal volume at 60 °C for 1 h, then transferred to fresh liquid paraffin for 1 h twice at 60 °C and then kept in liquid paraffin overnight at 60 °C. Flies were transferred to a plastic petri dish with fresh liquid paraffin at 60 °C and then allowed to cool down. Paraffin blocks were made from this. Sections of 5-μm thickness were taken using a LEICA RM 2235 rotary microtome and then dried overnight on the slide warmer at 37 °C and then processed for hematoxylin and eosin staining.

Murrah buffalo testis was fixed in 4% paraformaldehyde (Sigma, Saint Louis, MO, USA), dehydrated and paraffin embedded. The 5-μm-thick serial sections were obtained by Leica RM 2235 rotary microtome. The sections were processed in APES-acetone (1:49), de-paraffinized and rehydrated. Then, the sections were incubated with 3% hydrogen peroxide in methanol, boiled in 10 mM sodium citrate buffer, and permeabilized in 1% Triton X-100. After blocking with 5% bovine serum albumin, the sections were incubated with the primary antibody (PHB at 1:100 dilution, GeneTex, Irvine, CA, USA, GTX124491; CAPZB at 1:100 dilution, GeneTex, GTX101686; TEKT2 at 1:50 dilution, Proteintech 13518-1-AP, Chicago, IL, USA) overnight at 4°C. Sections were then washed 3 times in phosphate buffer saline (PBS)-Tween-20, incubated with Biotin-labeled goat anti-rabbit IgG at 1:100 dilution, (Proteintech SA00001-2, USA) for 45 minutes at room temperature and then at 45 minutes at 37°C. Immunoreactive signal was detected using streptavidin-HRP and diaminobenzidine (DAB Map Kit, Ventana, Tucson, AZ, USA). The negative controls were generated by replacing the primary antibody with PBS. The sections were observed with an Olympus DP70 digital camera mounted on a Leica DMR microscope with Nomarski optics (Leica, Heerbrugg, Switzerland).

Eight centimeter pieces of all 38 tagged corals were preserved in 10% neutral-buffered formalin for 24–48 hrs and then washed and transferred to 70% ethanol for long-term storage. From these pieces, two sets of three to five polyps were dissected from each specimen for histological analysis.
Polyps were decalcified using RDO Rapid Decalcifier (Apex Engineering Products Corporation) until all spicules were dissolved. These samples were then dehydrated to 100% ethanol, using a graded ethanol series (10–20% increments), and cleared using toluene. Samples were then infiltrated in molten paraffin wax for 24 hrs and embedded using standard histological molds. Five micron sections were cut for both fecundity (serially every 90 microns) and for oocyte size analysis (five centrally located slides) on a Leica RM2235 rotary microtome. Sections were stained with Masson's Trichrome or Haemotoxylin - Eosin (H&E) using standard procedures, and permanently coverslipped. Each slide was examined under an Olympus CX21 microscope, with Moticam 5 camera (Motic Optical) attachment used to grab images. Images were analyzed using Image J (NIH).