Each incubation vial contained 12 islets in 1.0 ml KRB buffer solution and was treated with 95%O2 5%% CO2 to obtain constant pH and oxygenation.
Sections were deparaffinised and rehydrated before undergoing antigen retrieval under pressure in 0.01 M sodium citrate buffer and were treated for endogenous peroxidase activity.
Next, the slides were washed three times in 2× SSC buffer and were treated with 1 % pepsin in 10 mM HCl in a coplin jar at 37 °C for 10 min.
The slides were pretreated in a microwave (900 W/15 min in citrate buffer at pH 6.0) and were treated for 5 min with hydrogen peroxide (3% hydrogen peroxide in methanol).
Specimens of heart, eye and the associated infraorbital sinus, central nervous system, lungs, kidney, spleen, liver and gonads (ovaries and testes) were collected and placed in 10% buffered formalin during autopsy, and were treated using a routine histological technique and embedded in paraffin.
Slides were immersed in citric acid buffer (0.01 M, pH 6.0) and were treated in an 800 W microwave oven for 2 minutes and then at 200 W for 8 minutes, for antigen retrieval.
The extracted tumor cells were dissolved in a total volume of 190 μL digestion buffer (DNA tissue mini kit, Qiagen) and were treated with proteinase K overnight at 56°C.
A blank sample containing only buffer was treated in the same manner and subtracted from the collected data.
One third of the lysate in Passive Lysis Buffer was treated with 200 µl Trizol reagent and chloroform to extract RNA for reverse transcription (RT -PCR.
Briefly, 40 μL of the TTR variants at 4 μM (in GF buffer) was treated with 4 μL of glutaraldehyde (25% solution, Sigma) and incubated at room temperature for 5 min. The reactions were quenched by addition of 4 μL of 7% NaBH4 (freshly prepared in 0.1 M NaOH).
