Tumor tissues were fixed with 10% formalin overnight and then embedded with paraffin. The tissue was cut into 4 μm sections, and the sections were dewaxed for immunohistochemistry. H&E staining was performed using a commercial staining kit (ab245880, Abcam) according to the manufacturer's instructions. For IHC staining, the section was blocked with 5% BSA for 1 h and then incubated with anti-Ki-67 antibodies (1 : 1000, ab270650, abcam) overnight. After washing with TBST buffer, the section was soaked with 2 drops of SignalStain® Boost Detection Reagent (HRP, Rabbit #8114, Cell Signaling Technologies) for 30 min at room temperature. Signal development was performed for 5 min using SignalStain® Substrate (#8059, Cell Signaling Technologies). The images were captured using a phase-contrast light microscope (Leica, Germany).
Signalstain substrate
Manufactured by Cell Signaling Technology
SignalStain® substrate is a chemiluminescent substrate used for the detection of horseradish peroxidase (HRP) conjugated secondary antibodies in western blotting applications. It provides a sensitive and stable signal for the visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Mounting medium, by Cell Signaling Technology (5 mentions)
Ab245880, by Abcam (1 mentions)
Ab270650, by Abcam (1 mentions)
Phase contrast light microscope, by Leica (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)
6 protocols using signalstain substrate
1
Immunohistochemical Analysis of Tumor Proliferation
2
Immunohistochemical Staining of FFPE Tumor Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
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!