5 μm sections were evaluated with immunohistochemistry for Ki67, a marker of proliferation and ERα using standard protocols [32 (link)]. Commercial antibodies were used including rabbit anti-ERα (EMD Millipore, Cat# 06–935, Temecula, CA, diluted 1:1000) and rabbit anti-Ki67 (Fisher Scientific, Cat# RM-9106-S1, diluted 1:1000). Secondary antibody (goat anti-rabbit, Abcam, Cat# ab64256) was followed by streptavidin peroxidase complex (Abcam, Cat# ab64269) and diaminobenzidene chromogen (Abcam, Cat# ab64238) to visualize reactions. Samples were counterstained with hematoxylin and coverslipped with permanent mounting media. Images were collected using a Zeiss Oberserver.Z1 inverted microscope with a 40x objective and Zeiss high-resolution color camera. Expression of each marker was determined by counting either mesenchymal or epithelial cells in each sample as indicated in the text.
High resolution color camera
Manufactured by Zeiss
Sourced in Germany
The high-resolution color camera is a precision instrument designed for accurate color imaging. It captures detailed, high-quality images with a focus on color fidelity and resolution.
Automatically generated - may contain errors
Lab products found in correlation
Axioimager dissection microscope, by Zeiss (2 mentions)
Oberserver z1 inverted microscope, by Zeiss (1 mentions)
Paraffin, by Leica (1 mentions)
Positively charged slides, by Thermo Fisher Scientific (1 mentions)
Rotary microtome, by Thermo Fisher Scientific (1 mentions)
Permanent mounting media, by Thermo Fisher Scientific (1 mentions)
Axio observer z1 inverted microscope, by Zeiss (1 mentions)
Zen pro software, by Zeiss (1 mentions)
Methyl salicylate, by Thermo Fisher Scientific (1 mentions)
Zen software, by Zeiss (1 mentions)
7 protocols using high resolution color camera
1
Immunohistochemical Analysis of Proliferation and Estrogen Receptor Expression
2
Paraffin Embedding and Histological Analysis
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Fixed mammary glands were washed in ×1 phosphate buffered saline, dehydrated through a series of alcohols and embedded with paraffin (Leica Biosystems, Richmond, IL) under vacuum. paraffin blocks were cut into five micrometer sections on a rotary microtome (Fisher Scientific) and mounted on positively charged slides (Fisher Scientific) for histological evaluation and immunohistochemistry.
For histological evaluations, slides were deparaffinized with xylene and a series of alcohols, stained with Harris’ hematoxylin and eosin (Fisher Scientific), dehydrated, and mounted with permanent mounting media (Fisher Scientific). Digital images were collected using a Zeiss Axio Observer. Z1 inverted microscope, a ×10 objective, and a high-resolution color camera (Carl Zeiss Microscopy).
For quantification of periductal collagen fibers, sections were stained with Gomori’s Trichrome Stain (Richard-Allen Scientific/Thermo Fisher Scientific, REF 87020). In short, deparaffinized samples were fixed with Bouin’s fluid (REF# 88038) and stained with Weigert’s Iron Hematoxylin (REF#s 88028 and 88029) and Gomori’s Trichrome Stain (REF# 88030). Individual dyes in the kit stain the collagen fibers blue, cell nuclei are stained black, while cytoplasm and muscle fibers appear red.
For histological evaluations, slides were deparaffinized with xylene and a series of alcohols, stained with Harris’ hematoxylin and eosin (Fisher Scientific), dehydrated, and mounted with permanent mounting media (Fisher Scientific). Digital images were collected using a Zeiss Axio Observer. Z1 inverted microscope, a ×10 objective, and a high-resolution color camera (Carl Zeiss Microscopy).
For quantification of periductal collagen fibers, sections were stained with Gomori’s Trichrome Stain (Richard-Allen Scientific/Thermo Fisher Scientific, REF 87020). In short, deparaffinized samples were fixed with Bouin’s fluid (REF# 88038) and stained with Weigert’s Iron Hematoxylin (REF#s 88028 and 88029) and Gomori’s Trichrome Stain (REF# 88030). Individual dyes in the kit stain the collagen fibers blue, cell nuclei are stained black, while cytoplasm and muscle fibers appear red.
3
Fetal Mammary Gland Histology Protocol
4
Apoptosis Detection in Ovarian and Uterine Tissue
5
Mammary Gland Morphometric Analysis
6
Mammary Gland Morphometric Analysis
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Whole mounts mammary glands were viewed and imaged using a Zeiss Axio Imager dissection microscope and Zeiss high-resolution color camera (Carl Zeiss, Oberkochen, Germany). Zeiss ZEN Pro software was used for morphometric analysis using methods developed previously [29 (link), 33 (link)]. Briefly, in females at both ages, specific measurements were quantified including the area subtended by the ducts (ductal area), the growth of the longest duct from the center of the lymph node (ductal extension), the total number of terminal end buds (TEBs, defined as bulb-shaped structures ≥0.03 mm2), and area of TEBs. Ductal density was evaluated using the threshold tool to compare intensity of mammary epithelium within 1.5 cm2 areas in the nipple and lateral to the lymph node, or across the entire ductal tree. In males, endpoints that were measured included ductal area and number of branching points. TEBs were never observed in adult males.
7
Quantification of Mammary Gland Ductal Density
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After fixation, whole-mounted mammary glands were processed through an alcohol series, defatted with toluene, stained with carmine alum, dehydrated in an alcohol and xylene series, and preserved in k-pax heat-sealed bags (Thermo Fisher Scientific) with methyl salicylate (Acros Organics, Morris Plains, NJ) [51 (link)]. Two digital images of whole-mount mammary glands (one from the left; one from the right third pectoral glands) were obtained using an AxioImager dissection microscope (Carl Zeiss Microscopy, Jena, Germany) at ×30 magnification and a Zeiss high-resolution color camera. To evaluate mammary gland morphology, ZEN software (Carl Zeiss Microscopy) was used to measure ductal density [51 (link)]. A 13 × 16 grid (180 crosshairs, 0.3 mm apart) was placed on each image, and the fraction of crosshairs that fell on ducts was quantified and averaged for the two images for each sample.
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