Bound fatty acids were transmethylated with NaOME (250 µl, sodium methoxide in dry methanol 0,5 M) by boiling at 45°C for 5 minutes.
Eluted proteins were purified by methanol precipitation, denatured by boiling in SDS buffer, and analysed by SDS/PAGE.
The presence of alkaloids was determined by first dissolving 0.02 g of extract in 1 mL methanol and filtered, followed by boiling the extract with 2 mL of 1% hydrochloric acid for 5 minutes.
Endogenous peroxidases were quenched in 0.3% H2O2 in methanol solution (30 min) followed by boiling (15 min) in a 10 nM sodium citrate solution (pH 6.0) for antigen retrieval.
Four-µm sections of formalin-fixed, paraffin-embedded tissues were deparaffinized, endogenous peroxidase was quenched with 0.3% H2O2 in methanol for 20 minutes, and antigen retrieval was performed by boiling the sections for 10 minutes in Tris-EDTA buffer (pH 9.0).
Eluted protein fractions were concentrated by methanol precipitation and pellets extracted into SDS dissociation buffer by boiling for 5 min.
After 15 min of incubation in 3%H2O22 in methanol, antigen retrieval (only in THG staining) was performed by boiling the slides for 15 min in a 0.01 M sodium citrate buffer (using a microwave oven).
For immunohistochemical staining, tissue sections were deparaffinized, rehydrated, incubated with 3%H2O22 in methanol for 15 min, and subjected to heat-induced antigen retrieval by boiling for 10 min in 0.01 M citric acid.
After blocking endogenous peroxidase activity in 0.5% H2O2 (100 volumes) in methanol for 35 min at room temperature, antigen retrieval was performed by boiling in 10 m citric acid buffer, pH 6.0, for 15 min in a microwave oven operating at full power.
To make the fatty acids volatile at ∼200°C (for separation by gas chromatography), the lipid extracts (25 ul) were transmethylated by boiling in excess (500 ul) boron trifluoride (BF3, in methanol 14% w/v) in a boiling water bath for 2 3 min. Methyl esters of long-chain fatty acids were extracted using 1.5 ml hexane/water (3∶2, v/v).
