The slides containing the maxilla sections were submerged in a citric acid-based antigen unmasking solution (Vector Laboratories) at 65°C overnight for antigen retrieval and then incubated with anti-CD68 antibody (cat. no. ab125212; Abcam) or p65 (cat. no. ab32536; Abcam) as primary antibodies. The tissue sections were visualized using 3-amino-9-ethylcarbazole (AEC; cat. no. SK-4205; Vector Laboratories, Inc.) at room temperature for 1 min, followed by counterstaining with hematoxylin for 1 min at room temperature, then sealed with ImmunoHistoMount™ solution (Agilent Technologies, Inc.). The digital images of the stained sections were obtained using the DP72 light microscope (Olympus Corporation).
Dp72 light microscope
Manufactured by Olympus
Sourced in Japan
The DP72 is a light microscope designed for high-quality imaging. It features a high-resolution digital camera and is capable of capturing detailed images of microscopic samples. The DP72 is a versatile instrument suitable for use in a variety of laboratory settings.
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12 protocols using dp72 light microscope
1
Immunohistochemical Analysis of Maxilla Sections
2
Morphological and Molecular Analysis of Pollen Development
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The morphological characteristics of grown plants were analyzed and the leaves were placed into mixture of ethanol and acetone (ratio 1:1) to detect absolute pigment content. The quantities of chlorophyll a (Chla), chlorophyll b (Chlb), Car and total Chl were calculated as the previous method described [78 ]. Pollen grains from anthesis buds of MT and WT were sampled, dissolved into mixed acids (chromic acid/nitric acid/hydrochloric acid, 15/10/5, v/v/v) and then stained by 2% iodine/potassium iodide solution (I2-KI). Flower buds of different lengths were collected in formalin-aceto-alcohol (FAA) for section observation to identify the pollen developmental stages and asses the cytological characteristics of MT. They were then photographed using an Olympus DP72 light microscope. Based on this identification, the anthers of the corresponding periods are selected for SEM and TEM. The detailed processes of paraffin sectioning, SEM and TEM were performed following the method described by Liu et al. (2015) [6 (link)]. Additionally, anthers from MT and WT during tetrad stage (bud, ~ 4.5 mm), late uninucleate stage (bud, ~ 8 mm), binucleate stage (bud, ~ 11 mm) were collected, frozen in liquid nitrogen immediately, and then stored at − 80 °C for RNA-seq and real-time PCR analysis.
3
Quantitative Immunohistochemical Analysis of BPH
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Descriptive statistics of the clinical parameters of 104 BPH patients are presented in Table 3 . Tissue from each of the 104 patient cases was fixed, made into donor wax block sections, stained with H&E for pathological diagnosis and lesion tissue localization, and evaluated and confirmed by senior pathologists. A 1.5 mm diameter core was taken from each sample wax block. Finally, we obtained tissue cores from all BPH samples, and the resulting TMA was then serially cut into sections with a thickness of 4 μm. In brief, paraffin sections were dewaxed and placed in citrate buffer (pH 6.0) for antigen repair, followed by blocking of endogenous peroxidase activity in 0.3% H2O2. Next, the above sections were incubated with the corresponding primary and secondary antibodies (listed in Supplementary Tables S3 and S4 , respectively). Antibody localization was identified by addition of peroxidase and 3, 3′-diaminobenzidine tetrahydrochloride. An Olympus DP72 light microscope (Olympus, Japan) was used to image stained sections. Two pathologists blinded to sample type quantified the expression of PPARγ, WNT-1, and β-catenin in prostate tissues derived from the TMA. Image J was used to measure the percentage of positive area for the three proteins.
4
Tissue Sectioning and Staining Protocol
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Those samples fixed in FAA for 24 h initially were sectioned longitudinally and transversely to a thickness of approximately 8 μm using a microtome. Then, the slices were positioned on the slides. These slides were finally treated with the following reagents in sequence: 1.0% safranin solution, 50% ethanol, 70% ethanol, 85% ethanol, 0.1% solid green solution, 100% ethanol, and resin sealing. These were finally inspected with an Olympus DP 72 light microscope.
5
Comprehensive Analysis of Plant Morphology and Cytology
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The morphological characteristics of grown plants were analyzed and the leaves were placed into mixture of ethanol and acetone (ratio 1:1) to detect absolute pigment content. The quantities of chlorophyll a (Chla), chlorophyll b (Chlb), Car and total Chl were calculated as the previous method described [78] . Pollen grains from anthesis buds of MT and WT were sampled, dissolved into mixed acids (chromic acid/nitric acid/hydrochloric acid, 15/10/5, v/v/v) and then stained by 2% iodine/potassium iodide solution (I 2 -KI). Flower buds of different lengths were collected in formalin-aceto-alcohol (FAA) for section observation to identify the pollen developmental stages and asses the cytological characteristics of MT. They were then photographed using an Olympus DP72 light microscope. Based on this identification, the anthers of the corresponding periods are selected for SEM and TEM. The detailed processes of paraffin sectioning, SEM and TEM were performed following the method described by Liu et al. (2015) [6] . Additionally, anthers from MT and WT during tetrad stage (bud, ~4.5mm), late uninucleate stage (bud, ~8mm), binucleate stage (bud, ~11mm) were collected, frozen in liquid nitrogen immediately, and then stored at -80°C for RNA-seq and real-time PCR analysis.
6
Comprehensive Pollen Development Analysis
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The morphological characteristics of grown plants were analyzed and the leaves were placed into mixture of ethanol and acetone (ratio 1:1) to detect absolute pigment content. The quantities of chlorophyll a (Chla), chlorophyll b (Chlb), Car and total Chl were calculated as the previous method described [78] .
Pollen grains from anthesis buds of MT and WT were sampled, dissolved into mixed acids (chromic acid/nitric acid/hydrochloric acid, 15/10/5, v/v/v) and then stained by 2% iodine/potassium iodide solution (I 2 -KI). Flower buds of different lengths were collected in formalin-aceto-alcohol (FAA) for section observation to identify the pollen developmental stages and asses the cytological characteristics of MT.
They were then photographed using an Olympus DP72 light microscope. Based on this identi cation, the anthers of the corresponding periods are selected for SEM and TEM. The detailed processes of para n sectioning, SEM and TEM were performed following the method described by Liu et al. (2015) [6]. Additionally, anthers from MT and WT during tetrad stage (bud, ~4.5mm), late uninucleate stage (bud, ~8mm), binucleate stage (bud, ~11mm) were collected, frozen in liquid nitrogen immediately, and then stored at -80°C for RNA-seq and real-time PCR analysis.
Pollen grains from anthesis buds of MT and WT were sampled, dissolved into mixed acids (chromic acid/nitric acid/hydrochloric acid, 15/10/5, v/v/v) and then stained by 2% iodine/potassium iodide solution (I 2 -KI). Flower buds of different lengths were collected in formalin-aceto-alcohol (FAA) for section observation to identify the pollen developmental stages and asses the cytological characteristics of MT.
They were then photographed using an Olympus DP72 light microscope. Based on this identi cation, the anthers of the corresponding periods are selected for SEM and TEM. The detailed processes of para n sectioning, SEM and TEM were performed following the method described by Liu et al. (2015) [6]. Additionally, anthers from MT and WT during tetrad stage (bud, ~4.5mm), late uninucleate stage (bud, ~8mm), binucleate stage (bud, ~11mm) were collected, frozen in liquid nitrogen immediately, and then stored at -80°C for RNA-seq and real-time PCR analysis.
7
Immunohistochemical Analysis of GRP78 in BPH
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The paraffin sections were deparaffinized in xylene prior to anhydrous ethanol, 95% alcohol and 75% alcohol in turn. They were subsequently kept in 10 mM boiled sodium citrate buffer (pH 6.0) for 2 min for antigen retrieval and incubated with 3% H2O2 solution for 10 min to inactivate endogenous peroxidase. To block non-specific binding, 15% normal goat serum was used to incubate sections for 15 min at room temperature. Next, the sections were incubated successively with GRP78 primary antibody (Table S7 ) at humidified and 4 °C conditions and with secondary antibody (Table S8 ) at 37 °C until peroxidase and 3, 3′-diaminobenzidine tetrahydrochloride visualization. Negative controls were incubated with PBS instead of the antibody. All stained sections were imaged using an Olympus-DP72 light microscope (Olympus, Japan). GRP78 expression in the prostate tissues from our TMA was blindly quantified by two pathologists via analysis for positive area of all images with Image J. For further correlation analysis, the clinical data were collected as we mentioned above. Pearson correlation analysis was conducted to investigate the correlation between GRP78 and several clinical characteristics of BPH, as well as GRP78 and multiple markers for apoptosis, EMT and OS.
8
BPH Tissue Microarray Analysis
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The summarized clinical data of 104 BPH patients was present in Supplementary Table S1 . The paraffin‐embedded specimens were sliced followed by hematoxylin and eosin (H&E) staining. The representative areas of the H&E staining sections were evaluated and confirmed by a senior pathologist. A TMA marker was designed by using 1.5 mm tissue core in each case. Finally, TMA contained 16 × 10 tissue cores for all BPH specimens in each were obtained and then sliced continuously into 4‐μm‐thick sections. Briefly, paraffin sections were deparaffinized first, then antigen retrieval was performed in citrate buffer (pH 6.0), and endogenous peroxidase activity was blocked in 0.3% H2O2. Subsequently, all slides were incubated with primary and secondary antibodies (listed in Supplementary Tables S2 and S3 ) until visualization by peroxidase and 3, 3′-diaminobenzidine tetrahydrochloride. All the stained sections were imaged using Olympus-DP72 light microscope (Olympus, Japan). The expression of SMO and GLI1–3 in the prostate tissues from the TMA was blindly quantified by two pathologists. The percentage of protein-positive area was measured by Image J.
9
Hematoxylin and Eosin Staining of Prostate Tissue
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Prostate paraffin sections (5 μm) were deparaffinized in xylene for 3 × 10 min, then rehydrated in descending concentrations of ethanol (100%, 96%, 80%, 70%) and H2O. The sections were then stained in 10% Hematoxylin (Sigma-Aldrich, St. Louis, USA) for 7 min, followed by washing under tap water for 10 min to reveal the nuclei. Afterwards, the sections were stained in 1% Eosin (Sigma-Aldrich, St. Louis, USA) containing 0.2% glacial acetic acid for 5 min. After staining, the sections were washed with tap water, dehydrated in increasing grades of ethanol (70%, 80%, 96%, 100%), and cleared in xylene for 3 × 10 min. The sections were imaged by an Olympus-DP72 light microscope (Olympus, Tokyo, Japan).
10
PDE5A Immunohistochemical Staining Protocol
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Sections for immunohistochemistry were deparaffinized in xylene followed by graded alcohols. Antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0) and heated to 96 °C for 3 min. Endogenous peroxidase activity was blocked by using 3% [v/v] H2O2 solution in methanol at room temperature for 10 min. Sections were incubated with 15% [v/v] normal goat serum for 1 h at 37 °C to block nonspecific binding. 100 μl appropriately diluted PDE5A primary antibody (1:100) was applied to the sections on the slides and incubated in a humidified chamber at 4 °C overnight. Then the sections were stained by routine immunohistochemistry methods. Negative controls were performed for all samples by omitting the primary antibodies. Rat lung tissue was used as a positive control for PDE5A staining. All the stained sections were imaged using Olympus-DP72 light microscope (Olympus, Japan).
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