Immunohistostaining of Ki67 was performed on 4-mm sections of formalin-fixed paraffin-embedded (FFPE) tumor tissue using VENTANA BenchMark Special Stain platform (Roche, Indianapolis, IN, USA). Briefly, the section was dehydrated and antigen unmasking was performed by heating the section submersed in 1X citrate unmasking solution (SignalStain® Citrate Unmasking Solution, Cell Signaling Technologies, #14,746) for 10 min at a sub-boiling temperature (95°-98°C). After cooling, sections were washed in dH2O three times and then incubated in 3% hydrogen peroxide for 10 min. The section was blocked 1 h at room temperature in TBST buffer with 5% Normal Goat Serum, and then incubated with primary antibody anti-Ki-67 (D3B5) (Cell Signaling Technologies, #9129) diluted 1:500 in SignalStain® Antibody Diluent (#8112) overnight at 4°C. After 3 washes with TBST buffer, the section was incubated with HRP-conjugated secondary antibody (1:3000 dilution; Cell Signaling Technologies, #7074) for 60 min at room temperature. After wash, the section was stained by a diaminobenzidine staining kit (ZSGB-BIO, Beijing, China) for 30 min at room temperature. The section was washed in dH2O two times and then dehydrated. Section was mounted with coverslips using the mounting medium (Cell Signaling Technologies, #14,177) before imaging.
Mounting medium
Manufactured by Cell Signaling Technology
Mounting medium is a laboratory reagent used to mount and preserve tissue samples or cells on microscope slides. It creates a transparent, protective layer over the sample, allowing for long-term storage and visualization.
Automatically generated - may contain errors
Lab products found in correlation
Signalstain substrate, by Cell Signaling Technology (5 mentions)
Diaminobenzidine staining kit, by ZSGB-BIO (1 mentions)
Signalstain citrate unmasking solution, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Anti ki 67 d3b5, by Cell Signaling Technology (1 mentions)
Ventana benchmark special stain platform, by Roche (1 mentions)
Signalstain antibody diluent, by Cell Signaling Technology (1 mentions)
Anti ki67, by Abcam (1 mentions)
Anti ki67 antibody, by Cell Signaling Technology (1 mentions)
Signalstain boost detection reagent, by Cell Signaling Technology (1 mentions)
Ab15580, by Abcam (1 mentions)
Goat serum, by Merck Group (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Normal goat serum (ngs), by Cell Signaling Technology (1 mentions)
Lc3 antibody, by Cell Signaling Technology (1 mentions)
7 protocols using mounting medium
1
Immunohistostaining of Ki67 in FFPE Tumor Tissue
2
Immunohistochemical Staining of FFPE Tumor Tissue
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For IHC staining, 4-mm sections of formalin-fixed paraffin-embedded (FFPE) tumor tissue were first deparaffinized in xylene and dehydrated in ethanol series. The antigen unmasking was performed by heating the section in citrate unmasking solution (SignalStain® Citrate Unmasking Solution (10X) (#14,746), Cell Signaling Technologies) for 10 min at a sub-boiling temperature (95°–98°C). After cooling, sections were washed in dH2O three times for 5 min each and then incubated in 3% hydrogen peroxide for 10 min. After three times washes in TBST buffer, the section was blocked for 1 h in 5% normal goat serum, and then incubated with primary antibodies overnight at 4°C: anti-PAPPA (1:500) and anti-Ki-67 (1:1000) (all from Abcam, Cambridge, UK). Then antibody solution was discarded and the section was washed three times using TBST buffer. The section was soaked with 1–3 drops SignalStain® Boost Detection Reagent (HRP, Rabbit #8114, Cell Signaling Technologies) for 30 min at room temperature. 200 µl SignalStain® substrate (#8059, Cell Signaling Technologies) was added for signal development. After 10 min, the section was washed in dH2O two times and then dehydrated. Section was mounted using the mounting medium (#14,177, Cell Signaling Technologies) and was imaged under Leica AM6000 microscope.
3
Immunohistochemical Analysis of Tissue Samples
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Tissue samples from patients or animal models were fixed with 4% formaldehyde overnight and then embedded in paraffin. Tissues were cut into 10-μm sections before staining. The tissue section was deparaffinized and hydrated, and the antigen unmasking was performed by heating the section in 1× citrate unmasking solution [SignalStain Citrate Unmasking Solution (10×), #14746; Cell Signaling Technology] at 95°C for 10 min. Sections were cooled for 30 min and washed three times in TBST buffer. After blocking for 1 h in TBST with 5% normal goat serum, the section was incubated with primary antibody anti-Ki67 antibody (1:500 dilution, #S9129; Cell Signaling Technology) and anti-SRGN antibody (1:500 dilution, #sc-374657; Santa Cruz) overnight at 4°C. The section was washed three times using TBST buffer and then soaked with 1–3 drops of SignalStain Boost Detection Reagent (HRP, Rabbit #8114; Cell Signaling Technology) for 30 min at room temperature. Then, 200 µl SignalStain substrate (#8059; Cell Signaling Technology) was added to each section for 5 min. The section was washed in distilled water twice for 5 min each and then dehydrated. The section was then mounted with coverslips using a mounting medium (#14177; Cell Signaling Technology) before imaging.
4
Immunohistochemical Staining of Paraffin Sections
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Paraffin-embedded tissues were cut into 4-5 μm sections using a microtome. After de-paraffinization and hydration, antigen retrieval was conducted by boiling the slides in citrate buffer for 90 s, followed by the incubation with 3% hydrogen peroxide for 10 min. After 3 washes in TBST buffer, the sections were blocked for 1 h in TBST with 5% normal goat serum, and then probed with primary antibodies (1:500) overnight at 4 °C. Following washing with TBST buffer, the sections was soaked with 1–3 drops of SignalStain® Boost Detection Reagent (Cell Signaling Technologies, Inc.) and incubated in a humidified chamber for 30 min at room temperature. The signal was developed using 400 μl SignalStain® substrate (Cell Signaling Technologies, Inc.) for 5 min. The sections were mounted with coverslip using mounting medium (Cell Signaling Technologies, Inc.) before being imaged under a bright-field microscope [23 (link), 24 (link)].
5
Immunohistochemical Analysis of FFPE Tissues
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Immunohistostaining was performed on 5‐μm sections of formalin‐fixed paraffin‐embedded (FFPE) tissues. The sections were deparaffinized and hydrated by three washes of xylene for 5 min each, two washes of 100% ethanol for 10 min each, two washes of 95% ethanol for 10 min each, and in two washes in dH2O for 5 min each. After antigen retrieval with citrate buffer at 95°C for 10 min, the sections were washed in dH2O three times and then incubated in 3% hydrogen peroxide for 10 min. After three times washes in TBST buffer for 5 min, the section was blocked for 1 h using 10% goat serum (Sigma, Germany) at 37°C, followed by the incubation with primary antibodies (HIP1R: Abcam ab226197; Ki67: Abcam ab15580) overnight at 4°C. Next day, the slides were washed 3 times with PBS and incubated with SignalStain® Boost Detection Reagent (HRP Rabbit, Cell Signaling Technologies) and incubated in a humidified chamber for 30 min. The signal development was performed for 5 min using 400 μl SignalStain® substrate (Cell Signaling Technologies). The section was washed in dH2O two times and mounted with coverslips using the mounting medium (Cell Signaling Technologies) before imaging under an inverted microscope (Olympus). The IHC staining was examined and scored independently by two experienced pathologists.
6
Immunohistochemical Staining of Tumor Tissues
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The tumor tissues were dehydrated, paraffin-embedded, and cut into 5 µm thick slices. Further, the tissue sections were treated with pepsin for 10 min at room temperature. After washing with PBS, the endogenous peroxidase was inactivated with 3% H 2 O 2 water for 30 min. The tissue sections were blocked by adding 150 µL of goat serum solution at room temperature for 1 h, and incubating the primary antibody at 4 °C overnight. Then, 2-4 drops of HRP-conjugated secondary antibody were added to the tissues for 1 h incubation, which was followed by the addition of 400 µL Sig-nalStain® substrate (Cell Signaling Technologies). In the end, the cell nuclei were counterstained with hematoxylin for 5 min and the section was washed with double-distilled water for 15 min. Section was mounted with cover slips using the mounting medium (Cell Signaling Technologies) before imaging.
7
Immunohistochemical Analysis of LC3 in Euthanized Mice
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After the last behavior test, mice were performed with the euthanize via decapitation. Hippocampus tissues were isolated and embedded into paraffins after dehydration using ethanol. Tissues were cut into 3-µm-thickness sections. Sections was performed with Dewaxing and rehydration for IHC. Sections soaked in 1× Citrate Unmasking Solution (Cell Signaling Technology, Danvers, MA, USA) to be heated under microwave for 10 min at 95 °C after the solution boiling. After incubated in 3% H 2 O 2 for 10 min, sections were incubated with 1× TBST containing 5% normal goat serum (#5425, Cell Signaling Technology, Boston, MA, USA) at room temperature for 1 h. Sections were incubated with diluted LC3 antibody (#12741, 1:500, Cell Signaling Technology) at 4 °C overnight, followed by the culture with Boost Detection Reagent (Cell Signaling Technology) for 30 min at room temperature. Sections were stained by DAB solution for 5-10 min. Sections soaked in ddH 2 O were dehydrated by ethanol and sealed using Mounting Medium (Cell Signaling Technology). Numbers of the positive cells were determined under a microscope (Olympus, Tokyo, Japan).
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