1. COLORECTAL CANCER PLASMA, TUMOR TISSUE, NORMAL TISSUE 2013.04.22
Tu M2-PK Detection
COLORECTAL CANCER
PLASMA, TUMOR TISSUE, NORMAL TISSUE

2. Plan for Tu M2-PK study in colorectal cancer
Materials: 370 patients who have plasma, tumor tissue and correspondent normal tissue.
Methods:
For plasma: ELISA (Schebo. Biotech Company)
For tissue: Immunohistochemistry (Schebo. Biotech Company)

3. Tumor M2-PK - EDTA Plasma test

4. Tumor M2-PK Antibodies – Tissues test

5. M2-PK staining of a human colon carcinoma with the anti-M2-PK antibody clone DF4
Immunohistochemical Staining
Immunohistology, also known as "immunohistochemistry" is the use of antibodies to stain histological sections. 

6. Immunohistochemistry Vs immunofluorescence
This technique, immunohistochemistry (IHC), is methodologically identical to immunofluorescence (IF), which had been in use since 1941 (Coons et al., 1941). The increasing popularity of IHC over IF is due to four main factors. First, IF requires special equipment, a fluorescence microscope and a designated darkened area. IHC, on the other hand, can be performed using a standard light microscope. Second, IF requires a dark field, so it is not possible to examine fluorescence patterns and cell and tissue morphology simultaneously. IHC utilizes a light microscope for visualization, so antigen localization and cell and tissue morphology can be observed at the same time. Third, fluorochrome-based signals last only 3 to 10 days, depending on the signal intensity and storage conditions. The colored precipitate in IHC staining, particularly with 3,3¢-diamnobenzidine (Table ), remains vibrant for years, and provides an excellent permanent record. Fourth, IF fluorochromes are light sensitive, so special care must be taken throughout the staining procedure to minimize loss of signal. The enzymes and most substrates used in IHC, on the other hand, are relatively light insensitive, and therefore the staining procedure can easily be performed on the open benchtop without any unusual precautions. The disadvantage of some IHC chromogens is their known carcinogenic effects, but using gloves when handling these reagents is an adequate safety precaution. The availability of a variety of enzyme-conjugated antibodies and colored substrates (see Table ) has made IHC more convenient and accessible to many laboratories.

7. Ref.

8. SRSF2 cloning study
New primer designed:
Product size: 662 bp

9. PCR results SRSF2 –ex tag gradient [50-70 °C] Ex Tag 0.3
buffer Ex tag 5
dNTP 4
cDNA 5
Primer F 1
R 1 DW
Total 50
96  °C : 96  °C : grad °C : 72  °C : 20  °C
3min : 30 sec :30 sec : 5 min : --
35 cycles

10. PCR results SRSF2 – f-tag 58 °C annealing temp F-Tag 0.3
buffer F-tag 5
dNTP 1
cDNA 5
Primer F 1
R 1
DW 36.7
Total 50
IM9
ladder
500 bp
1000 bp
96  °C : 96  °C : 58°C : 72  °C : 20  °C
3min : 30 sec :30 sec : 5 min : --
35 cycles

11. PCR results SRSF2 – f-tag 60 °C annealing temp F-Tag 0.3
buffer F-tag 5
dNTP 1
cDNA 5
Primer F 1
R 1
DW 36.7
Total 50
K562 Hela IM9 ladder
96  °C : 96  °C : 60°C : 72  °C : 20  °C
3min : 30 sec :30 sec : 5 min : --
35 cycles