In this issue, we will present the 7-color staining results of human gallbladder cancer samples conducted in our laboratory. The experimental details are as follows:

Application of Multiplex Immunofluorescence(mIHC) Kit in Human Gallbladder Cancer

Sample: Human Gallbladder Cancer

Markers:  Epcam、vimentin、panck、ki67、CD31、CD45

mIHC Kit: NR11022, Nova 7-color Multi Fluorescence Staining Plus(mIHC) Kit (TYR-480/520/570/620/690/780 General secondary antibody for rabbits and mice）

   The main advantages of the two-step kit are reflected in the following two aspects: Firstly, it exhibits minimal non-        specific reactions, effectively reducing background signal interference, thereby ensuring clearer and more reliable results.  Secondly, the RC780 two-step method does not produce spectral overlap with the 690 channel, which means that in multi-channel experiments, data from each channel can remain more independent, avoiding analytical errors caused by signal crossover.

The experimental design for this study is outlined as follows:

TYR480-EpCAM

TYR520-Vimentin

TYR570-PanCK

TYR620-ki67

TYR690-CD31

TYR780-CD45

The scanning platform used in this application is from the ThermoFisher EVOS S1000 spatial imaging system.

The EVOS S1000 Spatial Imaging System simplifies the workflow for generating multiplex immunofluorescence images of tissue samples, enabling rapid understanding of protein spatial localization. The EVOS S1000 system can simultaneously image up to 8 targets plus nuclear staining in a single scan, utilizing real-time spectral unmixing to enhance resolution of the protein targets under investigation, thereby addressing fluorescence bleed-through issues between different channels. With the ease of use and precision of the EVOS S1000 system, high-quality data can be acquired quickly and in greater quantities.

Multiple fluorescence detection of 9 dyes (without using cyclic detection method)

EVOS S1000 System Advantages

• Fast – Generates a fully resolved, stitched 9-color fluorescence image of 1 cm² tissue area with a 20x objective within one hour

• Versatile – Capable of multispectral fluorescence, transmitted brightfield, phase contrast, and color brightfield imaging

• Sensitivity – High resolution with a 4.2-megapixel, 16-bit CMOS camera

• Adjustable Magnification – 5-position turret for switching between 2.5x to 40x objectives

• Efficiency – Laser- and software-based autofocus procedures streamline the image acquisition workflow

• User-Friendly – Intuitive software makes image collection, navigation, and data retrieval accessible to users of all experience levels

• Flexible – Over 30 dyes included in the Invitrogen EVOS S1000 software (compatible with primary antibody conjugates and spatial amplification reagents)

• Data Quality – Automatically generated quality metric reports help ensure high-quality image capture and provide actionable recommendations for improvements when needed.

Results display: 

                                                                                                                                        Tumor region segmentation based on DAPI+PanCK

         EVOS S1000 scanning+spectral analysis                                                            Segmentation result diagram: Red represents the PanCK region,

                                                                                                                                       green represents the stromal region, and yellow indicates blank areas.

Quantification of intra-organ vascularization                                        Cell proximity analysis                                          Cell infiltration analysis                                                                   

 EpCAM+/PanCK+/Ki67 co-expression

        EpCAM+PanCK                          EpCAM+PanCK+Ki67                        EpCAM+Ki67

Products information in this case:

For more information, please contact us.