Product Composition: Sodium- CITRATE pH6.0.
Intended Use: Product for in vitro diagnostic use
Recommendations for Use:
Before use: The CITRATE solution is 10x concentrated and therefore it must be diluted in a 1:10 range with distilled or deionized water (one part CITRATE buffer and 9 parts water).
1. – Cut and mount sections on either charged or silane coated slides.
2A. – In case of using an automated PT Module for simultaneous dewaxing, rehydrating and antigen (HIER) retrieval, follow the instructions enclosed in the equipment data sheet. Basically, fill the tanks with 1.5 litres of diluted, ready-to-use buffer solution, place the slides without dewaxing into the racks and then immerse them into the buffer solution. Program the PT Module for a cycle of antigen retrieval of 20min at 95ºC and then cool to room temperature. Then follow with the step 4.
2B. – In case of not using the PT Module for HIER continue with dewaxing (using xylene or substitute) and rehydration of the tissue sections through decreasing series of ethanol. Then follow with the step 3 and 4.
3. – Unmasking: different alternative methods could be used to carry out the antigen retrieval
process using the diluted CITRATE buffer:
3.1 Unmasking in microwave: Place the sections in the diluted solution of CITRATE (Coplin jar or other container with a volume as large as the laboratory considers) and bring the microwave oven to 700 watts (watt) for 10-15 minutes). Cool to room temperature.
3.2 Unmasking in pressure cooker: Fill the pressure cooker with 1-2 litres of diluted CITRATE buffer. While hydrating the slides, heat the solution avoiding boiling. Then immerse the sections in the preheated buffer and close the pressure cooker. Wait until the pressure rises to the second notch and run from here 1-2 minutes (depending on the antigen unmasking). Remove and cool the pot (you can speed up the process placing the pot under running water). Open when there is no pressure and cool the slides inside of the pot for 20-30 minutes.
4. – Remove the sections and place them into the usual washing buffer (recommended TBS / TBS-Tween pH 7.5 – reference MAD-004077R)
5. – Proceed with the immunostaining protocol as usual.