Bx40 f 3 microscope

Paraffin-embedded specimens of endometrium from endometriosis and control groups, were sectioned at 7μm, prepared with xylene and ethanol, pressurized and heated with proteinase K (Welgene, Daegu, Korea) for antigen retrieval. Endogenous peroxidase activity was quenched with 3% H₂O₂ for 10 minutes and washed in buffered saline solution (PBS; Welgene, Daegu, Korea) for 5 minutes twice. Unspecified binding of the first antibody was blocked by 1 hour incubation step in 5% bovine serum albumin (BSA; Gibco, Invitrogen, Carlsbad, CA, USA). Slides were incubated in a humidified chamber overnight at 4°C with HMGB-1 rabbit polyclonal antibody at 1:100 dilution (ProteinTech, Chicago, IL, USA). Tissue sections were washed with PBS, tris-buffered saline (TBS; Gibco, Invitrogen) was used for antibody dilutions and washes. Sections were incubated with rabbit polyclonal secondary antibodies (Vector Laboratories, Burlingame, CA, USA) for 20 min at room temperature. Diaminobenzidine was used as a chromogen at 1 mg/mL, with 0.3% H₂O₂ as a substrate. All sections were counterstained with hematoxylin for 1 min and washed with tap water. After dehydration, mounting was performed. Slides were documented using an Olympus BX 40F-3 microscope (Olympus, Tokyo, Japan). Intensities of immunoreactive precipitates were scored in epithelial and stromal cell compartments using Olympus DP controller software.

A 1-cm ileal specimen per bird was prepared as the procedure described by Jiang et al. (2020) (link). Briefly, the formalin-fixed samples were dehydrated in graded ethanol solutions from 70 to 100%, cleared with xylene, then embedded in paraffin. Thereafter, 5.0-µm thick sections were sliced using a Leica RM 2145 microtome (Leica, Nussloch, Germany). The sections were stained with hematoxylin and eosin (Thermo, Waltham, MA), then examined using an Olympus BX40F-3 microscope (Olympus Cooperation, Tokyo, Japan). Three tissue sections containing intact lamina propria were selected from each bird, and an average of two readings (villus height, VH and crypt depth, CD, both measured in μm) were made from each section (total 6 counts per bird, 42 counts per group per time point). Image J software (NIH, Bethesda, MD) was used to measure VH and CD. The VH and CD per tissue sample were averaged, and the VH/CD ratio was calculated.

Five intestinal samples were randomly selected from each group and processed according to the method described by Thompson et al. [29 (link)]. In brief, the intestinal samples were collected from the distal (about 1 cm from the colon). Intestinal samples were dehydrated with increasing concentrations of ethanol, cleared with xylene (Surgipath Medical Industries, Richmond, IL), and embedded in paraffin wax (Thermo Fisher Scientific, Kalamazoo, MC). Cross-Sect. (5 μm) were stained with hematoxylin and eosin (GeneCopoeia, Rockville, MD). The stained sections were dehydrated with ethanol, cleared with xylene, and mounted with DPX mountant based on the method of Jiang et al. [30 (link)]. ImageJ software (National Institutes of Health, USA) was used to determine the morphometric measurements of villus height and crypt depth of the cecal using an Olympus BX40 F-3 microscope (Olympus Cooperation, Tokyo, Japan) attached to a digital video camera (Q-imaging, 01-MBF-200R-CLR-12, SN: Q32316, Canada) [31 (link)].

The histomorphologal parameters of the jejunal tissue samples were measured using previously published methods [34 (link), 38 (link)]. Briefly, the jejunal tissue samples were dehydrated and embedded with paraffin wax (Thermo fisher scientific, Kalamazoo, MC). The paraffin blocks were cut at 5-μm-thick cross sections using a microtome, then stained with hematoxylin and eosin (H&E) (GeneCopoeia, Rockville, MD), and examined under an Olympus BX40 F-3 microscope (Olympus Cooperation, Tokyo, Japan) attached to a digital video camera (Q- imaging, 01-MBF-200R-CLR-12, SN: Q32316, Canada) as described in Jiang et al. [34 (link)]. The morphometric measurements of villus height and crypt depth of the jejunum were analyzed by using the software of Image J (National Institutes of Health, USA).