Simple Procedure with High Sensitivity

The protocol is generally as simple as the secondary antibody method. After labeling the primary antibody, just add the β-galactosidase-labeled secondary antibody and the staining solution, then the sample is ready for detection.

Cell Line: HeLa Cells
Antigen: CD44
Instrument:
(Left) Fluorescent microscope Ex = 340 - 380 nm, Em = 435 - 485 nm
(Right) Flow Cytometer　Ex = 405 nm, Em = 425 - 575 nm

Application Data: PD-L1 Expressed HepG2 Cells

  PD-L1 expressed HepG2 cells and CFSE-stained control cells were prepared and mixed. The results showed the CLAMP stained cells did not localize in the CFSE stained cells, indicating that the CLAMP method accurately differentiates PD-L1 expressed HepG2 cells, which was difficult using the secondary antibody method.
*CFSE: 5- or 6-(N-Succinimidyloxycarbonyl)fluorescein 3‘,6’-diacetate

＜General Protocol＞

・For adherent cells

＜Experimental Conditions＞
　Blue (CLAMP): Ex=340-380 nm, Em=435-485 nm
　Green (CFSE): Ex=450-490 nm, Em=500-550 nm

　Even in situations where cells were adjacent to each other, only PD-L1-expressing cells were stained, and no misstaining of other cells was observed.

・Single Cell Analysis [SIEVEWELL^TM (TOKYO OHKA KOGYO Co., LTD.)]

scale bar: 100 μm

SIEVEWELL^TM enables isolating cells into single cell and culturing on microwells. Combining the CLAMP staining method with SIEVEWELL^TM, target cells can be separated accurately. After separation on the SIEVEWELL^TM, picking rare cells and more precise cell separation are expected.

For details, please refer to the web page of TOKYO OHKA KOGYO Co., Ltd. https://www.sievewell.com/how-it-works

Application Data: FFPE Tissue Sections - Human Small Intestine

Using the CLAMP method and TSA (Tyramide signal amplification) method detected the αSMA (α-smooth muscle actin) and keratin on FFPE tissue sections of human small intestine samples. The results showed that the CLAMP method can easily co-stain with immunofluorescence. Please refer to the reference No.1 for a detailed protocol on the CLAMP method for tissue sections.

* The data was kindly provided by Dr. Masahiro Hirata, Department of Diagnostic Pathology, Kyoto University Hospital.

Application Data for each protocols in the manual

Protocol 1(Application Data: CD44 Antigen in HeLa Cells)

In accordance with protocol 1 of the manual, the CD44 antigen of live HeLa cells was detected using the CLAMP method. As a result, a clear difference in fluorescence intensity between the control and the stained samples was observed, indicating that the surface antigen can be detected with high sensitivity.

＜Protocol＞
1. Inoculated HeLa cells (1 x 10⁵ cell/ml) into a 96-well microplate(100 μl/well) and incubated in a CO₂ incubator at 37 ℃ overnight.
2. Discarded the supernatant and washed the cells with 100 μl of MEM (10% FBS) twice.
3. Added 100 μl of 2 μg/ml Anti-CD44 antibody (ab189524, abcam)/MEM (10% FBS) and incubated at 37℃ for 60 minutes.
4. Discarded the supernatant and washed the cells with 100 μl of MEM (10% FBS) twice.
5. Added 100 μl of x500 diluted β-Galactosidase-labeled Anti-Rabbit IgG/MEM (10% FBS) and incubated at 37℃ for 60 minutes.
6. Discarded the supernatant and washed the cells with 100 μl of HBSS twice.
7. Added 100 μl of Staining Solution and incubated at 37℃ for 30 minutes.
8. Discarded the supernatant and washed the cells with 100 μl of HBSS three times.
9. Observed the cells under a fluorescence microscope.（DAPI Filter: Ex = 320-360 nm, Em = 435-480 nm）

Protocol 2 (Application Data: CD 44 antigen in HeLa cells by Flow Cytometer)

In accordance with protocol 2 of the manual, the CD44 antigen of HeLa cells was measured using a flow cytometer. We compared the CLAMP method with the secondary antibody method. As a result, the CLAMP method can detect the antigen with higher sensitivity by a flow cytometer.

＜Protocol＞
1. Prepare 2 x 10⁵ cells/tube of a cell suspension in a 1.5 ml microtube.
2. Centrifuge at 300 × g for 5 minutes and discard the supernatant.
3. Add 500 μl of 2 μg/ml Anti-CD44 antibody（ab189524, abcam）/MEM medium（10% FBS）and mix by pipetting.
4. Incubate at 37℃ for 60 minutes.
5. Perform the following washing step:
    I. Centrifuge at 300 x g for 5 minutes and discard the supernatant.
    II. Add 500 μl of medium and mix by pipetting.
    III. Centrifuge at 300 x g for 5 minutes and discard the supernatant.
    IV. Perform steps II and III again.
6. Add 500 μl of x500 β-Galactosidase labeled antibody (ab16774, abcam) /MEM medium (10% FBS), and mix by pipetting. (Secondary antibody method used Alexa Fluor405 antibody (ab175652, abcam))
7. Incubate at 37℃ for 60 minutes.
8. Perform the same washing step as in step 5 with HBSS. (Used HBSS instead of medium)
9. Add 500 μl of Staining Solution (Floating) and mix by pipetting.
10. Incubate at 37℃ for 30 minutes with stirring using a tube rotator. (RVM-101, Scinics)
11. Centrifuge at 300 x g for 5 minutes and discard the supernatant.
12. Add 500 μl of HBSS and mix by pipetting.
13. Measure by a flow cytometer. (LSR Fortessa X-20、BD)
*BV421 filter set (Ex:405 nm, Em:430-470 nm)

Protocol 3 (Application Data: Anti-CD44 antigen in fixed HeLa Cells)

In accordance with protocol 3 of the manual, the CD44 antigen of fixed HeLa cells was detected using CLAMP method under live-cell experimental conditions. As a result, a clear difference in fluorescence intensity between the unstained sample and the fixed sample was observed, indicating that surface antigens can be detected with high sensitivity.

＜Protocol＞

Preparing PFA fixed cells
1. Inoculate HeLa cells (1 x 10⁵ cell/ml) into a microplate and incubate in a CO₂ incubator at 37 ℃ overnight.。
2. Discard the supernatant and wash the cells with 100 μl of PBS twice.
3. Add 100 μl of 4% PFA/PBS and incubate at room temperature for 30 minutes.　
4. Add 100 μl of 0.1% Triton-X100/PBS and incubate at room temperature for 30 minutes.
5. Add 100 μl of Blocking Buffer and incubate at room temperature for 30 minutes.
Staining Protocol
1. Add 100 μl of 2 μg/ml Anti-CD44 antibody (ab189524, abcam) /Blocking Buffer and incubate at room temperature for 60 minutes.
2. Discard the supernatant and wash the cells with 100 μl of PBS twice.
3. Add 100 μl x500 β-Galactosidase labeled antibody (ab16774, abcam) /Blocking Buffer and incubate at room temperature
for 60 minutes.
4. Discard the supernatant and wash the cells with 100 μl of PBS twice.
5. Add 100 μl of the Staining Solution and incubate at 37℃ for 30 minutes.
6. Discard the supernatant and wash the cells with 100 μl of PBS three times.
7. Observe the cells under a fluorescence microscope.（DAPI filter: Ex = 320-360 nm, Em = 435-480 nm）

Protocol 4 (Application Data: PD-1 antigen in fixed MOLT4 Cells)

In accordance with protocol 2 of the manual, the PD1 antigen in fixed MOLT4 cells was detected using a flow cytometer by CLAMP method and secondary antibody method. The results showed that the CLAMP method had a higher sensitivity than the secondary antibody method with flow cytometer.

＜Protocol＞
Preparing fixed cells
1. Prepared MOLT4 cells (2 x 10⁵ cells/tube) of a cell suspension in a 1.5 ml microtube.
2. Centrifuged at 300 × g for 5 minutes and discarded the supernatant.
3. Added 500 μl of 4% PFA/PBS and mixed by pipetting.
4. Incubated at room temperature for 30 minutes, then centrifuged at 300 × g for 5 minutes.
5. Discarded the supernatant, added 500 μl of 0.1% Triton-X100/PBS, and mixed by pipetting.
6. Incubated at room temperature for 30 minutes, then centrifuged at 300 x g for 5 minutes.
7. Discarded the supernatant, added 500 μl of Blocking Buffer (10% Blocking One/PBS), and mixed by pipetting
8. Incubated at room temperature for 30 minutes, then centrifuged at 300 x g for 5 minutes, Used the fixed cells for the staining process.
Staining protocol
1. Add 500 μl of 1 μg/ml mouse Anti-PD1 antibody（ab52587, abcam）/Blocking Buffer and mix by pipetting.
2. Incubate at room temperature for 60 minutes.
3. Perform the following washing step:
　I. Centrifuge at 300 x g for 5 minutes and discard the supernatant.
　II. Add 500 μl of Blocking Buffer and mix by pipetting.
　III. Centrifuge at 300 x g for 5 minutes and discard the supernatant.
　IV. Perform steps II and III again.
4. Add 500 μl of X3,000 β-Galactosidase-labeled Anti-mouse antibody（ab136775, abcam) /Blocking Solution and mix by pipetting.
5. Incubate at room temperature for 60 minutes.
6. Perform the same washing step as in step 3.
7. Add 500 μl of Staining Solution (Floating ) and mix by pipetting。
8. Incubate at 37℃ for 30 minutes with stirring using a tube rotator.
9. Centrifuge at 300 x g for 5 minutes and discard the supernatant.
10. Add 500 μl of Blocking Buffer and mix by pipetting.
11. Measure by a flow cytometer.（LSR Fortessa X-20, BD）
*BV421 filter set (Ex:405 nm, Em:430-470 nm) 

Protocol 5 (Application Data: Actin in frozen tissue section of mouse liver)

In accordance with protocol 5 of the manual, the CLAMP method without primary antibody was compared with the normal CLAMP method. As a result, the CLAMP method was found to be highly sensitive and selective for Actin staining.

＜Protocol＞
PFA Fixation
1. Bring a frozen tissue section on a slide glass to room temperature.
2. Draw a border around the tissue section using a water-repellent pen.
3. Add 100 μl of 4% PFA/PBS and incubate at room temperature for 30 minutes.　
4. Discard the supernatant, add 100 μl of 0.1% Triton-X100/PBS and incubate at room temperature for 30 minutes.
5. Discard the supernatant, add 100 μl of Blocking Buffer and incubate at room temperature for 30 minutes.
Staining Protocol
1. Discard the supernatant, add 100 μl of 2 μg/ml Anti-actin antibody (GTX109639, GeneTex)/Blocking Buffer and incubate at room temperature for 60 minutes.
2. Discard the supernatant and wash with Blocking Buffer twice.
3. Add 100 μl of x500 β-Galactosidase labeled Antibody (ab16774, abcam)/Blocking Buffer and incubate at room temperature for 60 minutes.。
4. Discard the supernatant and wash with Blocking Buffer twice.
5. Add 100 μl of the Staining Solution and incubate at 37℃ for 30 minutes.
6. Discard the supernatant and wash with PBS three times.
7. Observe the cells under a fluorescence microscope.（DAPI filter: Ex = 320-360 nm, Em = 435-480 nm）

Protocol 6 (Application Data: Actin in FFPE tissue sections of mouse liver)

In accordance with protocol 6 of the manual, Actin in mouse liver FFPE tissue sections was measured using fluorescence microscopy. The CLAMP method without primary antibody was compared with the conventional CLAMP method. As a result, the CLAMP method was found to be highly sensitive and selective for Actin staining.

＜Protocol＞
Antigen Activation
1. Prepare FFPE tissue section on a slide glass.
2. Incubate at 60℃ for 60 minutes.
3. Incubate at room temperature for 15 minutes.
　  Note: Use a humidity chamber not dry the sections during the following processes.
4. Soak the slide with lemosol for 5 minutes. Repeat this step.
5. Soak the slide with ethanol for 5 minutes.
6. Soak the slide with 90% ethanol aqueous solution for 5 minutes.
7. Soak the silde with 80% ethanol aqueous solution for 5 minutes.
8. Wash the slide by soaking in ultrapure water, followed by PBS.
9. Soak the slide with antigen activation solution (pH=9), and heated to 95℃ for 40 minutes.
10. Incubate at room temperature for 60 minutes.。
11. Wash the slide by soaking in ultrapure water, followed by PBS.
Staining protocol
1. Draw a border around the tissue section using a water-repellent pen.
2. Add 100 μl of Blocking Buffer and incubate at room temperature for 30 minutes.
3. Discard the supernatant, add 100 μl of 2 μg/ml Anti-actin antibody (GTX109639, GeneTex) /Blocking Buffer and incubate at room temperature for 60 minutes.
4. Discard the supernatant, add 100 μl of PBS and incubate at room temperature for 3 minutes. Repeat this step twice.
5. Add 100 μl of  x500 β-Galactosidase labeled secondary antibody (ab16774, abcam社) /Blocking Buffer and incubate at room temperature for 60 minutes.
6. Discard the supernatant, add 100 μl of PBS and incubate at room temperature for 3 minutes. Repeat this step twice.
7. Add 100 μl of Staining Solution and incubate at 37℃ for 30 minutes.
8. Discard the supernatant, add 100 μl of PBS and incubate at room temperature for 3 minutes. Repeat this step twice.
9. Observe the cells under a fluorescence microscope.