Zenon alexa fluor 594

Paraffin sections of the SMG (4-μm thick) were deparaffinized in xylene and rehydrated in graded alcohol series. Sections were immersed in citrate buffer pH 6.0 (Epitomics) at 97°C for 20 min, washed in PBS and immersed in peroxidase blocking (DAKO) for 10 min. The samples were then washed in PBS and incubated in protein block (DAKO) for 1 h. The first antibody phosphorylated ERK (1:150, Cell Signaling Technology, Cat# 4370, RRID:AB_2315112) was prepared with Zenon Alexa Fluor 594 (Molecular Probes, Inc., Eugene, OR) according to manufacturer’s instructions, and applied to the samples and incubated in a humidified dark chamber for 2 h. Then the sections were washed in PBS for 25 min and subsequently incubated with the second antibody phosphorylated CREB (1:150, Abcam Cat# ab32096, RRID:AB_731734) prepared with Zenon Alexa Fluor 488 for 2 h. DAPI was used for nuclear counterstaining.

Paraffin sections of the SMGs (4 μm thick) were deparaffinized in xylene and rehydrated in a graded alcohol series. Sections were immersed in citrate buffer pH 6.0 (Abcam, Inc., Cambridge, MA, EUA) at 97°C for 20 min, washed in PBS and immersed in peroxidase blocking solution (DAKO) for 10 min. The samples were then washed in PBS and incubated in protein block solution (DAKO) for 1 h. The first antibody Bax (1:150, Abcam) was prepared with Zenon Alexa Fluor 594 (Molecular Probes, Eugene, OR, USA) according to the manufacturer’s instructions, applied to the samples and incubated in a humidified dark chamber for 2 h. The sections were then washed in PBS for 25 min and were subsequently incubated with the secondary antibody to HMGB1 (1:150, Abcam) prepared with Zenon Alexa Fluor 488 for 2 h. DAPI was used for nuclear counterstaining.