Antibodies Plant/Algal / DNA/RNA/Cell Cycle / Nuclear signaling

Immunostaining using anti-hydroxy guanosine monoclonal antibody

Tissue Preparation
Anti-hydroxy guanosine antibody  monoclonal antibody reacts on both 50 µm frozen tissue sections and paraffin-embedded sections. Tissue should be dissected fresh and fixed in periodate-lysineparaformaldehyde (PLP) at 4°C
overnight.

PLP
Heat 1 L dest. water to 60°C. Add 60 g paraformaldehyde. Add 33 g dibasic NaPO4. Cool to room termperature in a cold water bath. Add 9 g monobasic NaPO4
.
Add 6.45 g Na-m-periodate.
Add 41.1 g lysine (HCl salt).
Filter and dilute to 3 L with dest. water. Adjust pH to 7.6 with 1.0 N NaOH
approx. (20-30 ml).
Tissue prepared for frozen sectioning must be cryoprotected in a 20% glycerol-2% DMSO solution in phosphate buffer for 24-48 hours. Tissue will sink to the bottom of container when fully penetrated. This will eliminate freezing artifact from cutting.

Glycerol-DMSO (for 3 L)
2.4 L 0.1 M phosphate buffer
600 ml glycerol
60 ml DMSO

0.1 M Phosphate Buffer, pH 7.4 (for 1 L)
1 L dest. water
11 g dibasic NaPO4
3 g monobasic NaPO4
After frozen sectioning, tissue should be stored in phosphate buffer with 0.08% sodium azide.

Staining Sections By DAB Procedure
Paraffin-embedded sections must be deparaffinized by sequential immersion in the following for 3 minutes each: xylene (twice), absolute ethanol (twice). Agitate gently in each solution. Proceed with the following procedure:
1. Pretreat sections with a methanolperoxide solution to eliminate endogenous peroxidases.

Methanol-Peroxide
100 ml absolute methanol
1 ml 33% H2O2
Incubate sections in methanolperoxide
solution for 30 minutes, room temperature.
2. Wash sections 3 times for 10 minutes each in 0.1 M phosphate buffered saline (PBS)
PBS, pH 7.4 (for 1 L)
1 L dest. water
11 g dibasic NaPO4
3 g monobasic NaPO4
8.5 g NaCl
3. Incubate sections for 1 hour in 10% normal goat serum in PBS.
4. Incubate sections in the primary antibody for 18-24 hours at room temperature. Depending on the nature
of the sample, a shorter incubation time may be used.