Human tissue, including the normal breast tissue and tumor tissue, were obtained with the pre-approval of the Institutional Review Board at the Xuanwu Hospital, Beijing, China. Invasive-ductal-carcinoma samples were obtained from patients undergoing surgical removal while the pathologically normal breast tissue were obtained 2 cm away from the tumor lesions in the same patient. Informed consent or assent was obtained from all patients and/or their parent or legal guardian as appropriate. To measure the nuclear volume within surgical tumor samples, these samples were sectioned and fixed with 4% paraformaldehyde for 1 hr. Then the central section crossing the tumor mass center was stained with Hoechst 33342 (Sigma, 14533) for another hour to visualize cell nuclei and washed 3 times with PBS. The 3D structure of the tumor slice was imaged with confocal microscopy and volume was calculated as described above. To exclude the lymphocytes for analysis, we labeled the pan-cytokeratin (a common epithelial cell maker for human, ThermoFisher, MA5–13156) in patient samples using antibodies and found that the tumor tissue indeed contains many pan-cytokeratin negative cells, suggestive of non-cancer cells (Fig. S13a ). However, we rarely find lymphocytes near the invasive acinar-like structures and almost all the cells within the structure are positive of pan-cytokeratin (Fig. S13b ).
Ma5 13156
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MA5-13156 is a laboratory instrument designed for protein and nucleic acid analysis. It is a precise and reliable tool that can be used in a variety of applications within the field of life sciences research.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse 2000 microscope, by Nikon (3 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Ms 348 p, by Thermo Fisher Scientific (2 mentions)
Anti pan cytokeratin, by Thermo Fisher Scientific (1 mentions)
C6219, by Merck Group (1 mentions)
Axioimager apotome microscope, by Zeiss (1 mentions)
Ab124964, by Abcam (1 mentions)
6 protocols using ma5 13156
1
Measuring Tumor Nuclear Volume in Breast Cancer
2
Immunofluorescence Analysis of Endometrial Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunofluorescence (IF) analysis, tissue sections were processed as previously described [52 (link),80 (link)] and incubated with the following primary antibodies: polyclonal rabbit anti-FOXL2 (homemade [81 (link)], 1/300), anti-α-SMA (Abcam ab124964, 1/500, Cambridge, UK), anti-Cx43 (Sigma Aldrich C6219, 1/400, St. Louis, MO, USA), and monoclonal mouse anti-PCNA (Sigma Aldrich P8825, 1/500), anti-pan-cytokeratin (Thermo Fisher MA5-13156, 1/400, Waltham, MA, USA). Sections were then incubated with donkey or goat secondary antibodies conjugated with Alexa Fluor 555, 488, or 649 (Thermo Fisher, 1/1000), followed by Hoechst staining. IF images were captured with a Zeiss AxioImager apotome microscope (Carl Zeiss Microscopy, Jena, Germany) at the IGH Imaging facility (BioCampus) and processed with the OMERO software (OMERO web 5.5.1., University of Dundee and Open Microscopy Environment, Dundee, UK). The intensity of α-SMA staining was determined in regions of interest (ROIs) (n = 4–12 ROIs per section) of a constant surface (30 mm2) at the endometrial stroma-junctional zone (2–3 sections of each uterus, n = 4–7 per group). Cx43 staining intensity was determined in ROIs (n = 4–10 ROIs per section, n = 3 per group) in the endometrial stroma (1500 μm2), luminal epithelium (1000 μm2), and glandular epithelium (700 μm2) (OMERO software). Data were analyzed with GraphPrism 7.
3
Immunohistochemical Profiling of Epithelial Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression of additional epithelial markers such as transmembrane mucin 1 (MUC1), also known as epithelial membrane antigen (EMA), and cytokeratin cocktail (AE1/AE3) were detected by immunohistochemistry methods. Cells were fixed with 4% paraformaldehyde in PBS 1X, treated with 0.1% Triton x-100 in PBS for 15 min and blocked with 2% BSA in PBS at RT for 2 h. Then, primary anti-EMA antibodies (MS-348-P, Thermo Scientific, USA) and anti-AE1/AE3 (MA5–13156, Thermo Scientific, USA) were incubated with cells at 4 °C for 16 h at 1:100 dilution. Finally, the slides were washed in PBS and incubated with an HRP conjugated goat anti-mouse IgG secondary antibody at RT for 45 min. Immunocytochemical staining was performed using an avidin-biotin complex peroxidase standard staining kit. HeLa and AGS cell lines (ATCC CRL-1739) were used as positive controls for AE1/AE3 and MUC1 markers, respectively, whereas hematopoietic U-937 cell lines (ATCC® CRL-1593.2™) were used as a negative control. Imaging was made using a Nikon Eclipse 2000 microscope (Nikon, Tokyo, Japan). All experiments were performed in triplicate.
4
Measuring Tumor Nuclear Volume in Breast Cancer
5
Immunohistochemical Characterization of Epithelial Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression of additional epithelial markers such as transmembrane mucin 1 (MUC1), also known as epithelial membrane antigen (EMA), and cytokeratin cocktail (AE1/AE3) were detected by immunohistochemistry methods. Cells were xed with 4% paraformaldehyde in PBS 1X, treated with 0.1% Triton x-100 in PBS for 15 min and blocked with 2% BSA in PBS at RT for 2 hours. Then, primary anti-EMA antibodies (MS-348-P, Thermo Scienti c, USA) and anti-AE1/AE3 (MA5-13156, Thermo Scienti c, USA) were incubated with cells at 4 o C for 16 hours at 1:100 dilution. Finally, the slides were washed in PBS and incubated with an HRP conjugated goat anti-mouse IgG secondary antibody at RT for 45 min. Immunocytochemical staining was performed using an avidin-biotin complex peroxidase standard staining kit. HeLa and AGS cell lines (ATCC CRL-1739) were used as positive controls for AE1/AE3 and MUC1 markers, respectively, whereas hematopoietic U-937 cell lines (ATCC ® CRL-1593.2 ™ ) were used as a negative control. Imaging was made using a Nikon Eclipse 2000 microscope (Nikon, Tokyo, Japan). All experiments were performed in triplicate.
6
Immunohistochemical Characterization of Epithelial Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression of additional epithelial markers such as transmembrane mucin 1 (MUC1), also known as epithelial membrane antigen (EMA), and cytokeratin cocktail (AE1/AE3) were detected by immunohistochemistry methods. Cells were xed with 4% paraformaldehyde in PBS 1X, treated with 0.1% Triton x-100 in PBS for 15 min and blocked with 2% BSA in PBS at RT for 2 hours. Then, primary anti-EMA antibodies (MS-348-P, Thermo Scienti c, USA) and anti-AE1/AE3 (MA5-13156, Thermo Scienti c, USA) were incubated with cells at 4 o C for 16 hours at 1:100 dilution. Finally, the slides were washed in PBS and incubated with an HRP conjugated goat anti-mouse IgG secondary antibody at RT for 45 min. Immunocytochemical staining was performed using an avidin-biotin complex peroxidase standard staining kit. HeLa and AGS cell lines (ATCC CRL-1739) were used as positive controls for AE1/AE3 and MUC1 markers, respectively, whereas hematopoietic U-937 cell lines (ATCC ® CRL-1593.2 ™ ) were used as a negative control. Imaging was made using a Nikon Eclipse 2000 microscope (Nikon, Tokyo, Japan). All experiments were performed in triplicate.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!