On Day 6 of culture, ovaries were fixed in Bouin's fluid and embedded in paraffin wax. Serial sections (5 µm thick) were collected and every sixth section stained with haematoxylin and eosin and photomicrographed (DMLB Leica microscope, Leica Microsystem Ltd, UK). Follicle counts were performed with the assessor blind to treatments and using ImageJ software. Total number of follicles per ovary was estimated from counts of follicles with a visible germinal vesicle in the section, with counts then corrected using the Abercrombie formula (Abercrombie, 1946) (link). Follicles were classified in accordance with Morgan et al. (2013) (link). Follicles were considered as primordial when only flattened pre-granulosa cells (GCs) were present, as transitional when some cuboidal GCs were mixed with flat pre-GCs and at the primary stage when a uniform layer of cuboidal GCs was present. Follicle health was assessed using standard morphological criteria: follicles were judged as unhealthy if they had (i) an oocyte with eosinophilic, shrunken or non-homogeneous cytoplasm, or condensed nuclear chromatin; (ii) GCs with condensed chromatin or irregular shapes; (iii) unhealthy oocyte and GCs.
Dmlb microscope
Manufactured by Leica
Sourced in Germany, United Kingdom, United States, Canada, Switzerland
The DMLB microscope is a high-quality optical instrument designed for laboratory use. It features a sturdy construction and precise optical components that enable clear, detailed observations. The DMLB microscope is capable of various magnification levels, allowing users to examine samples with precision.
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Lab products found in correlation
Dfc310 fx camera, by Leica (6 mentions)
Las ez software, by Leica (5 mentions)
Dab chromogen, by Agilent Technologies (5 mentions)
Dibutylphthalate polystyrene xylene (dpx), by Merck Group (4 mentions)
Qwin software, by Leica (4 mentions)
Mc170 hd camera, by Leica (3 mentions)
Image pro plus 7, by Media Cybernetics (3 mentions)
Rabbit anti mmp13, by Abcam (3 mentions)
Mouse anti col2α1, by Santa Cruz Biotechnology (3 mentions)
Rabbit anti adamts5, by Santa Cruz Biotechnology (3 mentions)
Mouse anti active β catenin, by Merck Group (3 mentions)
Rm 2155, by Leica (3 mentions)
Coolsnap pro, by Media Cybernetics (3 mentions)
Acetone, by Merck Group (3 mentions)
Powershot a95, by Leica (3 mentions)
Q500 iw, by Leica (3 mentions)
Ec3 digital camera, by Leica (3 mentions)
Bouin s solution, by Merck Group (2 mentions)
Naphthol as tr phosphate, by Merck Group (2 mentions)
L tartaric acid, by Merck Group (2 mentions)
222 protocols using dmlb microscope
1
Ovarian Follicle Enumeration and Classification
2
Ovarian Follicle Morphology Analysis
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Paraffin wax blocks were sectioned at 5 µm, and either taken for immunohistochemistry (IHC; details below) following fixation of tissue in Neutral Buffered Formalin (Sigma Aldrich), or photomicrographs taken of haematoxylin and eosin-stained sections (DMLB Leica microscope, Leica Microsystems Ltd) following fixation of tissue in Bouin’s solution (Sigma Aldrich), with stained sections used to undertake morphological examination of ovarian follicles.
3
Analyzing Root Cortical Cell Length
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At the end of growth monitoring, the root apices were fixed in 4% paraformaldehyde in a phosphate-buffered saline solution for 30 min under vacuum (n=4–5 roots per genotype or treatment). Fixed samples were rinsed three times with distilled water then dehydrated in a graded ethanol series and embedded in Technovit 7100 resin (Heraeus Külzer embedding kits) following the manufacturer’s instructions. Samples were stored at 35 °C for at least 4 d. Longitudinal sections of 5-μm thick were cut in the middle of the root with a rotary microtome (Microm HM355S, Thermo Scientific). The sections were stained with Toluidine Blue O, mounted in synthetic resin (Eukitt®) and examined under a DMLB Leica microscope equipped with a Leica DFC420C camera (Leica Microsystems). Longitudinal sections were imaged at 100× magnification. Images were assembled using MosaicJ (Thévenaz and Unser, 2007 (link)). Cortical cell length and its distance from the QC were measured semi-automatically from longitudinal sections using a macro in Fiji (Bizet et al., 2015 (link)). The cell length profile along the apex was interpolated with a cubic smoothing spline using R (smooth.spline, spar=0.9 to 1).
4
Histological Assessment of Seminiferous Tubules
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Wax blocks were sectioned at 5 µm, and either taken for IHC (see below) or photomicrographs taken of haematoxylin and eosin-stained sections (DMLB Leica microscope, Leica Microsystems Ltd, UK) and used for morphological examination by a blind-to-treatment assessor using ImageJ software. In each section, the number of seminiferous tubules that lacked visible germ cells on the basement membrane (Sertoli cell-only tubules) was noted, and the diameter of every spherical tubule was measured.
5
Quantifying Germ Cell Density and Apoptosis
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H&E stained sections were photomicrographed (DMLB Leica microscope, Leica Microsystems Ltd, UK), with images taken using a DFC369FX camera on a Leica DM5500B microscope (Leica Microsystem Ltd, UK), using filters for DAPI, A4 (BP 360/40); for Alexa Fluor 488, N3 (BP 546/12); for Alexa Fluor 568, Y5 (BP 620/60). Image J software was used for image analysis, with the assessor blind as to treatment. Germ cells (MVH + ) were analyzed by manual counting and the total number given relative to the area of seminiferous tubule, in order to obtain a value for germ cell density (number of cells that were MVH-positive divided by seminiferous tubule area in mm 2 ). Density was also determined for whole section area (number of MVH positive cells divided by the area of section in mm 2 ) to allow determination of germ cell density even where tubule integrity had been disrupted to the extent that precluded measurement of tubule area. For CC3, fluorophore area was measured as a percentage of tubule, interstitium and section area (proportion of CC3-positive area in mm 2 relative to area of seminiferous tubule, interstitium or whole section, all in mm 2 ).
6
Histological Analysis of Lamin A/C Mutant Mice
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Skeletal muscle or myocardium fragments from Lmna+/+ or LmnaG609G/G609Gmice were frozen in melting isopentane and stored in liquid nitrogen. Unfixed cryosections were subjected to immunofluorescence staining as detailed above.
Samples of aortic arch promptly after necropsy were fixed with formalin and embedded in paraffin. Histology was based on hematoxylin–eosin, PAS stain (Bio Optica, Milan, Italy; P.A.S. Hotchkiss – MC Manus, 04‐130802) and Alcian stains at pH 2,5 and 1 (Bio Optica; Alcian Blu, 04‐160802). Reduction of cellularity in aorta middle coat was graded as described (Zaghini et al.,2020 ) and reported in Table S1 . For aorta examination, three photographs per sample were acquired with a DFK 33UX264 camera coupled with a Leica TM DMLB microscope (TIFF format 2448 × 2048, Obj 20×).
The area was manually delineated (lowest selected area = 40,000 µm2), and leiomyocytes were manually counted with ImageJ software (https://imagej.nih.gov/ij/index.html/ ). Finally, cellularity was assessed and expressed as the number of leiomyocytes/10,000 µm2.
Samples of aortic arch promptly after necropsy were fixed with formalin and embedded in paraffin. Histology was based on hematoxylin–eosin, PAS stain (Bio Optica, Milan, Italy; P.A.S. Hotchkiss – MC Manus, 04‐130802) and Alcian stains at pH 2,5 and 1 (Bio Optica; Alcian Blu, 04‐160802). Reduction of cellularity in aorta middle coat was graded as described (Zaghini et al.,
The area was manually delineated (lowest selected area = 40,000 µm2), and leiomyocytes were manually counted with ImageJ software (
7
Quantitative Analysis of Paneth Cells in Intestinal Lamina Propria
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The quantitative analysis of PCs was conducted according to the method of Waly et al. (2001) (link). PC counts were performed in three anatomo-functional areas of the intestinal lamina propria: (1) the lamina propria of the villi (area 1), (2) the lamina propria of the upper part (area 2) and, (3) lower part (area 3) of the crypt (Fig. 1 ). For each area three to six photographs were randomly acquired with a Leica TM CCD camera DFC320 coupled with a Leica TM DMLB microscope (JPEG format, 2088 × 1550 pixel, Obj 40×). Then the lamina propria area was manually delineated to exclude large blood and lymphatic vessels, and the epithelium (lowest selected area = 4000 μ2). Finally PC density was assessed and expressed as the number of PCs/10,000 μ2. Digital image analysis was performed with ImageJ software (http://rsbweb.nih.gov/ij/ ).![]()
On the right, the reference areas proposed by Waly et al. (2001) (link). Anatomo-functional areas of the small intestine: area 1 (lamina propria of the villus); area 2 (lamina propria of the upper part of the crypt); area 3 (lamina propria of the deep part of the crypt).
8
Immunohistochemistry of Paraffin-Embedded Prostate
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Immunohistochemical assays of paraffin-embedded tissues sections were performed using the protocol described in Justulin et al.33 (link). Ventral prostates were fixed in 4% paraformaldehyde dissolved in phosphate buffer saline for 24 hours. Fixed samples were dehydrated in graded ethanol series, clarified in xylene and embedded in Paraplast (Sigma-CO). Five-µm prostate sections were cut and mounted on silanized slides. Prior to staining, the sections were dewaxed, rehydrated and then unmasked in 0.01 M citrate buffer (pH 6.0). The anti-CD34 (mouse polyclonal, sc74499, Santa Cruz Biotechnology), anti-Ki67 (rabbit monoclonal, ab16667, Abcam), anti-androgen receptor (rabbit polyclonal, sc816, Santa Cruz Biotechnology) antibodies were applied to the sections and incubated overnight at 4 °C. A secondary antibody complexed with peroxidase was utilized and the detection with 3,3′-diaminobenzidine as substrate was done. Counterstaining was performed with Harris hematoxylin. Analyzes were carried out using a DMLB Leica Microscope and the images were obtained by a Leica DFC300FX digital camera connected to the microscope.
9
Histological Analysis of Calvaria Bone
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Five‐micrometer‐thick deplastified calvaria bone sample sections were sequentially cleared in water and stained with toluidine blue (pH 3.8), von Kossa staining (n = 6 defects per group and per time point), or processed for ALP enzyme‐histochemistry and for tartrate‐resistant acid phosphatase (TRAP) revelation. Toluidine blue staining was used to visualize connective bone matrix. Von kossa staining was used to visualize mineralized bone. TRAP was detected by using hexazotized pararosanilin (Sigma, St. Louis, MO) and naphthol ASTR phosphate (Sigma) to reveal osteoclasts; nonosteoclastic acid phosphatase was inhibited by adding 100 mMol/l l (+)‐tartaric acid (Sigma) to the substrate solution. Image acquisition was performed using a DMLB Leica microscope, equipped with imaging camera DFC425 Leica connected to the Leica application (LAS version 4.4). The reconstruction of each field was made using Microsoft Image Composite Editor software (Microsoft, Redmond, WA, http://www.research.microsoft.com ), and image analysis was performed using ImageJ.
10
Histomorphometric Analysis of Calvaria Bone
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Sections (5 μm thick) of deplastified calvaria
bone samples
were sequentially stained with modified Masson–Goldner trichrome
or processed for alkaline phosphatase (ALP) and tartrate-resistant
acid phosphatase (TRAP) by enzyme histochemistry. Modified Masson–Goldner
trichrome staining allowed visualizing the osteoid and mineralized
bone tissues. TRAP was detected by hexazotized pararosanilin and naphthol
AS-TR phosphate (both from Sigma-Aldrich, St. Louis, MO) to reveal
osteoclasts; nonosteoclastic acid phosphatase was inhibited by adding
100 mMol/L of L(+)-tartaric acid (Sigma) to the substrate solution.
Image acquisition was performed using a DMLB Leica microscope, equipped
with an imaging camera DFC425 Leica connected to the Leica application
(LAS version 4.4).
bone samples
were sequentially stained with modified Masson–Goldner trichrome
or processed for alkaline phosphatase (ALP) and tartrate-resistant
acid phosphatase (TRAP) by enzyme histochemistry. Modified Masson–Goldner
trichrome staining allowed visualizing the osteoid and mineralized
bone tissues. TRAP was detected by hexazotized pararosanilin and naphthol
AS-TR phosphate (both from Sigma-Aldrich, St. Louis, MO) to reveal
osteoclasts; nonosteoclastic acid phosphatase was inhibited by adding
100 mMol/L of L(+)-tartaric acid (Sigma) to the substrate solution.
Image acquisition was performed using a DMLB Leica microscope, equipped
with an imaging camera DFC425 Leica connected to the Leica application
(LAS version 4.4).
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