Biopsy samples were fixed in 4% paraformaldehyde, dehydrated in ascending alcohol concentrations, and embedded in paraffin. Formalin-fixed, paraffin-embedded tissues were sequentially cut into 3 µm sections and mounted on positively coated slides. The histopathological evaluation of hematoxylin–eosin-stained sections was performed, and the mitotic count was calculated by counting mitotic events per 10HPF. Immunostains were performed using an automated stainer (Benchmark Ultra; Ventana Systems, Phoenix, AZ, USA). Antigen retrieval in Tris-based buffer (36 min at 95–100 °C) was followed by 32-min primary antibody incubation for mouse anti-human Ki-67 (clone MIB-1, Dako, Glostrup, Denmark) and 24-min incubation for mouse anti-human Chromogranin A (clone LK2H10, Merk KGaA, Darmstadt, Germany). An UltraView DAB detection kit (760–500, Ventana Systems, Phoenix, AZ, USA) was used in the detection reaction according to the manufacturer-recommended protocol. The hematoxylin counterstains were used for color development. The expression of the Ki-67 index protein was determined by calculating the ratio of positively stained nuclei among 100 tumor cells.
Mouse anti human ki 67 clone mib 1
Manufactured by Agilent Technologies
Sourced in Denmark, Japan, Germany
The Mouse anti-human Ki-67 (clone MIB-1) is an antibody that specifically binds to the Ki-67 protein, which is a nuclear protein associated with cellular proliferation. This antibody can be used to detect and quantify the expression of Ki-67 in human tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Pkh26, by Merck Group (1 mentions)
Fluoromount aqueous mounting medium, by Merck Group (1 mentions)
Zen 2009, by Zeiss (1 mentions)
Anti mouse alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Lsm 710, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Peroxidazed 1, by Biocare Medical (1 mentions)
Background sniper, by Biocare Medical (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Dab kit, by Vector Laboratories (1 mentions)
Vulcan fast red chromogen kit 2, by Biocare Medical (1 mentions)
Reveal decloaker, by Biocare Medical (1 mentions)
Rabbit anti granzyme b, by Abcam (1 mentions)
Rabbit anti t bet h 210, by Santa Cruz Biotechnology (1 mentions)
Bx43f microscope, by Olympus (1 mentions)
6 protocols using mouse anti human ki 67 clone mib 1
1
Immunohistochemical Analysis of Tumor Samples
2
Immunofluorescence Staining of Cell Surface Markers
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Rat anti-human/mouse CD44 (clone IM7; eBioscience Inc., San Diego, CA), rat anti-human CD44v9 (clone RV3; CosmoBio, Tokyo, Japan), and mouse anti-human Ki-67 (clone MIB-1; Dako, Glostrup, Denmark) antibodies were used for immunofluorescence staining. GM was purchased from Sigma-Aldrich (St. Louis, MO). 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE; Dojindo laboratories, Kumamoto, Japan), and PKH26 (Sigma-Aldrich, St. Louis, MO) were used for live-cell labeling.
3
Immunofluorescence Staining of Paraffin-Embedded Tissues
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The tissue samples were fixed in 4% paraformaldehyde and embedded in paraffin. Antigen retrieval of 3 μm thick deparaffinized sections was performed in 10 mM citrate buffer for 3 min in a pressure cooker. Sections were blocked with PBS/ 5% FCS for 20 min, afterwards the sections were stained with antibodies in PBS/ 5% FCS/ 0.1% Tween20 for minimum 45 min. Following antibodies were used: 4′,6-diamidino-2-phenylindole (DAPI) (Sigma); mouse anti-human-Ki67 (clone Mib-1, DAKO), anti-mouse-Alexa Fluor 546 (polyclonal goat, LifeTechnologies); anti-CD138-FITC (MI15, Biolegend). Sections were mounted with Fluoromount™ Aqueous Mounting Medium (Sigma-Aldrich). Confocal images were generated using a 20×/0.5 numerical aperture (NA) air objective lens on a Zeiss LSM710, provided with Zen 2010 Version 6.0 software. Images were analyzed using Zen 2009 or 2011 Light Edition software (Carl Zeiss MicroImaging).
4
Tryptase and Ki-67 Immunohistochemistry in Biopsies
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Biopsies were cross‐sectioned (5 μm thickness) and then deparaffinised, hydrated and boiled in a pressure cooker in Reveal Decloaker (Biocare Medical). Background sniper (Biocare Medical) was used to block non‐specific background staining. For tryptase staining, sections were incubated at room temperature for 2 h with a mouse monoclonal tryptase antibody (MAB1222, Chemicon Inc.) at 1/2000 dilution. Mouse AP polymer detection kit and Vulcan Fast Red Chromogen kit 2 (both from Biocare Medical) were used for visualisation. Sections were counterstained with Mayer's haematoxylin (Histolab). Incubation with mouse IgG was used as negative control. For Ki‐67 staining, endogenous peroxidase was blocked with Peroxidazed 1 (Biocare Medical) before incubation with the primary antibody: mouse anti‐human Ki‐67 clone MIB1 (Dako, A/S). As a secondary antibody, horse anti‐mouse biotinylated IgG (Vector Labs) was used. Vectastain ABC‐Elite kit and DAB kit (both from Vector Labs) were used for visualisation. Counterstaining was performed as above. Incubation with mouse IgG was used as negative control. Standard haematoxylin and eosin (H&E) staining was performed for histopathological evaluation.
5
Immunohistochemical Profiling of Tissue Samples
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The primary antibodies were as follows: goat anti-human CD105 (AF1097, R&D Systems), rabbit anti cyclooxygenase 2 (GTX15191, GeneTex), mouse anti-human CD68 (clone PG-M1, GeneTex), goat anti-CD3 epsilon (clone M-20, Santa Cruz Biotechnology), rabbit anti-T bet (H-210, Santa Cruz biotechnology), rat anti-human Foxp3 (PCH101, eBioscience), goat-anti proliferating cell nuclear antigen (PCNA) (clone C-20, Santa Cruz Biotechnology), mouse anti-human Ki-67 (clone MIB-1, Dakocytomation), rabbit anti-human CD8 (clone SP16, Thermo Fisher Scientific), mouse anti-human CD20 (clone L-26, Abcam), rabbit anti-human PD-L1 (Invitrogen, PA5-28115), rabbit anti-IL17 (H-132, Santa Cruz Biotechnology), mouse anti-human CD21 (clone 2G9, Thermo Fisher Scientific), rat anti-peripheral node addressin (clone MECA-79, BD Pharmigen), rabbit anti-human CD138 (RB-9422-P1, Thermo Fisher Scientific), mouse anti-podoplanin (clone D2-40, GTX31231, GeneTex), rabbit anti-granzyme B (clone EPR8260, Abcam), rat anti-human DC-LAMP (clone 1010E1.01, Novus Biologicals), rabbit anti-human prostate stem cell antigen (PSCA) (GTX15168, GeneTex), rabbit anti-human CXCL10 (GTX31176, GeneTex), biotin-mouse anti-smooth muscle actin (clone 1A4, Thermo Fisher Scientific), and mouse anti-human plasma cell (clone LIV3G11, Thermo Fisher Scientific).
6
Immunohistochemical Analysis of Tumor Markers
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Formalin-fixed, paraffin-embedded tumors were cut in 4 μm sections, de-paraffinized, and exposed to mouse anti-human Ki67 (clone MIB-1, Dako), rabbit monoclonal anti-human ERα (clone SP1, Dako), two different mouse anti-human ERβ, (clone PPG5/10 ERβ1 and clone 57/3 ERβ2, AbD Serotec, Puchheim, Germany), rabbit anti-human cleaved PARP (Abcam, Cambridge, UK) or rabbit anti-human von Willebrand factor (Dako) followed by anti-rabbit or anti-mouse HRP polymer kit (Dako). Sections were counterstained with Mayer's hematoxylin. Negative controls with omitted primary antibodies were included in each experiment and did not show any staining. All evaluation was performed in a blinded manner. Images of hot-spot areas of at least four tumors in each treatment group were acquired on an Olympus BX43F microscope (Lund, Sweden). The images were digitally analyzed and quantified using Olympus CellSens Dimension software version 1.5 (Hamburg, Germany). Clone 57/3 ERβ2 is shown in Supplementary Figure 2 .
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