Rm2245 rotary microtome

After the mature lamina joints of the wild-type and rela mutant were separated, they were rapidly fixed in formaldehyde-acetic acid-alcohol (FAA) at 4°C for 72 h. Subsequently, the samples were dehydrated through a gradient ethanol solution. The dehydrated samples were subjected to a xylene:ethanol clear solution of 1:2, 1:1, and 2:1 and finally transitioned to xylene. The samples were then incubated in a xylene:Paraplast Plus solution (Sigma–Aldrich) solution of 1:1 at 37°C for 48 h. The samples were embedded in Paraplast Plus for 3 days. The samples were then sectioned into 8-μm-thick sections by an RM2245 rotary microtome (Leica). After the removal of Paraplast Plus with xylene:ethanol ratios of 2:1, 1:1, and 1:2, the sections were rehydrated with gradient ethanol, followed by safranin-fixed green staining. Images were taken with an ICC50 HD microscope (Leica), and the cell length and cell area were subsequently measured using ImageJ.

After blood collection from the eyeball, mice were euthanized using cervical dislocation. Briefly, the hearts were rapidly excised and the heart weight-to-body weight ratio (HW/BW) was then calculated. Subsequently, the hearts were fixed in 4% tissue cell fixation solution for 24 h. Tissue sections (4 μm) were cut using a Leica RM-2245 rotary microtome and placed on polylysine-coated slides after dehydrated and embedded in paraffin. Routine hematoxylin and eosin (H&E) staining was performed using a staining machine (Leica Autostainer XL, ST5010). Digital scans (×40) of these slides were obtained using the Teksqray SQS-1000 slide scanning imaging system, and the images were visualized using ImageViewer (DPVIEW V2.0) software.

Three shrimps were collected per treatment and anesthetized with ice to collect the intestine, which was fixed in 10% formalin (NBF) for 24 h and later transferred to 70% alcohol. Samples of the anterior and middle intestines were processed in LEICA TP 1020 and included in the Paraplast. Intestines were sectioned into cross-sections at a thickness of 5 µm using a LEICA RM2245 rotary microtome and stained with hematoxylin–eosin (Harris hematoxylin), as suggested by Luna [40 ] and Romano and Pedrosa [41 (link)]. Histological slides were viewed under a Zeiss Primo Star optical photo microscope with an AxioCam ERc 5s camera (Göttingen, Germany). AxioVision LE 4.8.2 SP2 software (Göttingen, Germany) was used to analyze the photographs. The height, width, and area of intestinal microvilli were measured. Histological analysis was performed according to the protocol of Bullerwell et al. [42 (link)], in which four photos were taken per intestine (middle and posterior) of the shrimp, totaling 12 photos per treatment of each intestine. In each photograph, three microvilli were randomly selected to remove the established parameters. All measurements were performed using ImageJ Software 1.53.

Panicles were fixed in FAA fixative (50% ethanol, 10% formaldehyde, 5% acetic acid) overnight at 4 °C. After dehydration under a gradient series of ethanol and xylene, panicles were embedded in paraplast plus (Leica, Wetzlar, Germany) and sectioned to a thickness of 7 µm using the RM2245 rotary microtome. The paraffin sections were dewaxed in xylene and rehydrated in an ethanol series. The TUNEL assay was performed using the DeadEnd Fluorometric TUNEL system (Promega, Madison, MI, USA, #G3250), according to the manufacturer’s instructions. Signals were observed and imaged using an Imager D2 microscope (Carl Zeiss, Oberkochen, Germany).

Heart, kidney, lymph node, lungs, liver, and injection sites from the sacrificed mice were collected and fixed in 10% formalin for 48 h. The organs were further processed and embedded in paraffin, and 8-μm sections were cut using the Leica RM 2245 Rotary Microtome. The sections were then stained using the following hematoxylin–eosin staining procedure. Sections were first dewaxed in xylene (three changes of 2 min each) and then rehydrated in absolute alcohol (two changes of 2 min each), in 90% for 2 min, in 70% for 2 min, then washed in running tap water for 2 min and stained in hematoxylin for 3 min, and again washed in running tap water for 2 min. Sections were then washed in 70% alcohol for 2 min and stained in eosin for 3 min. Sections were then washed in 95% alcohol for 2 min and then in absolute alcohol (three changes of 2 min each). Finally, the sections were rapidly dehydrated and fixed in xylene (three changes of 2 min each) and mounted in DePeX. The sections were then observed under the Zeiss LSM 510 META confocal microscope at 20× magnification.

Heart, kidney, liver and injection sites from the sacrificed mice were collected and fixed in 10% formalin for 48 h. The organs were further processed and embedded in paraffin and 8 μm sections were cut using the Leica RM 2245 Rotary Microtome. The sections were then stained using the following haematoxylin and eosin staining procedure. Sections were first Dewaxed in xylene (3 changes of 2 min each), and then rehydrated in absolute alcohol (2 changes of 2 min each), in 90% for 2 min, in 70% for 2 min. Then washed in running tap water for 2 min and stained in haematoxylin for 3 min and again washed in running tap water for 2 min. Sections were then washed in 70% alcohol for 2 min and stained in eosin for 3 min. Sections were then washed in 95% alcohol for 2 min, then in absolute alcohol (3 changes of 2 min each). Finally, the sections were rapidly dehydrated and fixed in xylene (3 changes of 2 min each) and mounted in DePeX. The sections were then observed under the Zeiss LSM 510 META confocal microscope.

For the anatomical exam (light microscopy—LM), samples were dehydrated in an ethanol series, embedded in histological resin [65 ], and transversely and longitudinally sectioned with 3 or 3.5 μm thick in a RM 2245 rotary microtome (Leica, Wetzlar, Germany). The sections were stained with toluidine blue in phosphate buffer, pH 5.8 [66 (link)], and mounted. Observations and illustrations were obtained using a Leica DM 4500 B light microscope connected to a Leica DFC 320 digital camera. Scales were determined under the same optical conditions.
Samples embedded in historesin were stained with toluidine blue for the detection of phenolic compounds and mucilage [66 (link)]. Alcohol-stored and fresh material was free-hand sectioned and stained with period acid and Schiff (PAS) for neutral polysaccharides [67 ], with Sudan III and Sudan IV [68 ] for total lipids, and with Nadi reagent [69 ] for essential oils and oil-resin localization.