IHC of skin tissue was conducted using cyclin dependent kinase 1 (CDK1) (EPR165, Abcam, UK) and cyclin B1 (CCNB1) (Y106, Abcam, UK) antibodies for immunostaining. Skin sections (4 mm) were deparaffinized, rehydrated, blocked and then antigen-repaired. Then, the slides were incubated with rabbit polyclonal antibodies at a dilution of 1:350 at 37°C for 1 h and rinsed with PBS 3 times. Reaction enhancement solution was added, and the slides were incubated at room temperature for 30 min and rinsed with PBS 3 times. Then, the slides were incubated for 30 min with secondary antibodies (Rabbit antibody) at room temperature and rinsed with PBS three times. Finally, 3,3′-Diaminobenzidine was used to develop the color, after which the slides were redyed and sealed. The slides were viewed and photographed under a microscope (Motic, China). The magnification is ×200.The Image-Pro Plus 6.0 immunohistochemical image analysis system was used to analyze the collected images.
Epr165
Manufactured by Abcam
Sourced in United Kingdom
EPR165 is a monoclonal antibody that recognizes the EPR165 epitope. It can be used for the detection and quantification of the target protein in various applications, such as Western blotting and immunohistochemistry. The core function of EPR165 is to bind to and detect the specific target protein.
Automatically generated - may contain errors
3 protocols using epr165
1
Quantitative Immunohistochemistry of Skin Tissue
2
Immunohistochemical Analysis of CDK1 and MAD2L1
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The antibodies used for IHC consisted of anti-CDK1 rabbit monoclonal antibody (EPR165 [ab133327]; Abcam) and anti-MAD2L1 mouse monoclonal antibody (17D10 [ab10691]; Abcam). Slides with lengths of 1~2mm formalin-fixed, paraffin-embedded tissue sections from RMS were stained manually. For IHC staining, each slide was deparaffinized and rehydrated using xylene and then washed by a graded alcohol series and phosphate buffer saline (PBS). Antigen repair was performed by boiling the tissue slice in 0.01M citric acid buffer (pH6.0) for 15min and cooling it naturally to room temperature. Endogenous peroxidase was blocked using TBS/H2O2. The sections were incubated with first antibodies against human CDK1 and MAD2L1, and then incubated with goat secondary antibody against rabbit and mouse immunoglobulins (DAKO) at 37°C for 30 min.
3
Multimarker Analysis of Mouse Brain
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Brains were isolated from mice perfused transcardially with PBS and 2% PFA. Brains were cut sagittally and placed in 2% PFA overnight, subsequent to being placed in a gradient of sucrose solutions (10%, 20%, and 30%). Murine brains were frozen in optimal cutting temperature (O.C.T.) compound (Tissue-Tek, Tokyo Japan) in hexane pre-chilled in liquid nitrogen. Frozen brain sections (8-9 µm) were xed in methanol, rinsed in tris-buffered saline with 0.05% Tween 20 (TBST) and blocked with 10% FCS. Excess solution was shaken off before primary uorophore-conjugated antibodies (anti-CD11b: M1/70, Biolegend and anti-NS1: 4G4, Roy Hall, UQ) were applied. Regions of interest were selected based on immuno uorescent staining visualised on the Olympus BX-51 microscope using a DP-70 camera and Cell Sensor software. Slides were then incubated with metal-conjugated antibodies overnight at 4°C (anti-Ly6C (HK1.4, Biolegend), anti-CD86 (GL1, Becton Dickinson), anti-iba1 (EPR165, Abcam), anti-CD45, (30-F11, Biolegend), anti-FITC (FIT-22, DVS) and anti-NeuN (Fox3, Biolegend)). Iridium DNA intercalator (Fluidigm, 1:2000 in TBST) was applied to brain sections, prior to sections were rinsed in UltraPure water and left to air dry. If slides were not immediately ablated on the Hyperion, they were stored at 4°C until use.
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