Cx31 microscope

Anesthetized rats were perfused intracardially with phosphate buffer saline (PBS; pH 7.4) for 10 min, followed by buffered paraformaldehyde (PFA 4%, Sigma, St Louis, USA) for 10 min. The saline and PFA solutions were perfused at constant pressure of 95 mmHg. Pieces of the carotid external artery (3–4 mm length) were collected 2 mm rostral from the carotid sinus and post-fixed by immersion in buffered-PFA 4% for 12 h at 4°C. Carotid arteries were dehydrated in graded ethanol solutions followed by xylol, included in paraffin, sectioned at 5 μm and mounted on silanized slides. The vessels were stained with hematoxylin and eosin and photomicrographs were taken with an Olympus CX 31 microscope with a CCD camera (Olympus Corp, Japan). The internal diameter (ID) was measured from fixed tissues with the ImageJ software (NIH, USA).
Liver and kidney samples obtained from euthanized rats were fixed with 4% paraformaldehyde (Sigma, St Louis, USA), dehydrated and paraffin-embedded. Transverse sections (5 μM) were stained with Harris hematoxylin (5 min) and eosin (30 s) and mounted with Entellan (Merck, Whitehouse Station, NJ, USA). Microphotographs were obtained at 40x and 100x with and Olympus CX31 microscope (Olympus Corporation, Tokyo, Japan).

Four-micron-thick paraffin-embedded sections of CCA samples (n = 49) were prepared (23 (link)) and processed for immunohistochemistry analysis. Antibody against PCDH9 was purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA) (dilution, 1:100; R05779). A streptavidin-biotin kit (#KIT-9720; Maixin-Bio, Fuzhou, China) was used to visualize antibody binding to the tissue sections. Two individuals (GY and YC) independently evaluated all samples using an Olympus CX31 microscope (Olympus Optical). The final results were compiled using a semiquantitative scoring system as previously described (24 (link), 25 (link)).

Consecutive tissue sections (4 μm) of paraffin-embedded normal and tumor specimens were prepared and processed for immunohistochemical analysis as described previously [47 (link), 48 (link)]. Antibodies against pAMPKα (1:100), pACC (1:200), p-mTOR (1:50), pS6 (1:100), p4EBP1 (1:200), PTEN (1:100), MMP7 (1:100), and FN1 (1:150) were used to determine protein expression. Sections were scored blindly by two independent individuals using an Olympus CX31 microscope (Olympus Optical). The following values for scoring intensity were used: 0, absence of positive staining; 1, weak expression; 2, moderate expression; 3, strong expression. A mean percentage of positive tumor cells were determined in at least five fields of view at 400x magnification and assigned a value from 0 to 100%. The percentage of positive tumor cells and the staining intensity were multiplied to produce a weighted score for each case. The scores ranged from 0 (0% of cells staining) to 3 (100 × 3/100).

PRR11 expression in these 472 cases was evaluated by two individuals using an Olympus CX31 microscope (Olympus Optical). A semi-quantitative scoring system was used. In brief, staining intensity was assigned as follows: negative, 0; weak, 1; moderate, 2; and intense, 3. The percentage of immunoreactive cells was scored as 0∼1(0%∼100%). Theoretically, a weighted score ranging from 0 (0% of cells staining) to 3 (100% of the cells staining at 3+ intensity) was generated for each tissue core [20 (link)]. The final score for each tumor was calculated by averaging the score for two tissue cores. The case score > 0 was considered as positive. A four-tiered scale [0∼0.5, negative; 0.5∼1.5, weak (+); 1.5∼2.5, moderate (++); ≥;2.5, intense (+++)] was generated to grade the staining level of PRR11 for each case.

For histological analysis, epididymal fat was collected, fixed in 10% buffered formaldehyde and processed routinely for preparation of histological slides with paraffin sections 3 µm thick, stained with hematoxylin and eosin and mounted in resinous Entellan^® media (Merck Millipore, Darmstadt, Germany). Histomorphometric analyses were performed using optical microscopy employing a capture system and image analysis, which consists of a binocular Olympus CX31 microscope (Olympus Optical do Brazil Ltda, São Paulo, SP, Brazil) with an attached camera (SC30 CMOS Color Camera for Light Microscopy Olympus Optical do Brazil Ltda, São Paulo, SP, Brazil). Measurements were conducted using Image-Pro^® Express software (Targetware Informática Ltda of Brazil, Água Branca, SP, Brazil).

Consecutive sections (4 μm) of paraffin-embedded tissue microarrays blocks were prepared and processed for immunohistochemical analysis. The expression of SNAT1, p-mTOR, p-Akt, and VEGF proteins in the sections and in animal models was detected with appropriate antibodies against p-mTOR (dilution, 1:50; clone Y391; Abcam), SNAT1 (dilution, 1:100; clone S104-32; Abcam), p-Akt (dilution, 1:100; clone S473; Abcam), and VEGF (dilution, 1:200; clone SP28; Lab Vision). Two individuals evaluated the expression of these proteins using an Olympus CX31 microscope (Olympus Optical). A semiquantitative scoring system was used [30 (link)]. Specifically, underexpression was defined as no staining of tumor tissue or less positive staining than in matched normal tissue, normal expression was defined as positive staining positivity similar to that of matched normal tissue, and high expression was defined as significantly more positive staining than in normal tissue. Normal and negative staining were here defined as low SNAT1 expression. Staining was scored independently by 2 individuals who were blinded to each other’s findings. All conflicting calls on scoring were adjudicated by a third individual.

In the cingulum bundle (CB) and corpus callosum (CC), morphological alterations in myelin were observed and quantified. We obtained a total of 50 microscopic fields using a 40 × objective. Each area per field was previously calibrated at 19 700 μm². Images were acquired using a CX31 Olympus microscope equipped with a digital camera Infinity1 (Teledyne Lumenera®), and the Infinity Analyze® software (version 6.3.0).