Alexfluor antibody

A portion of the cardiac muscles were embedded in Tissue-Tek O.C.T. Compound (Sakura) and multiple thin sections (10 μm) were cut using a cryostat (Microm). Subsequently, the sections were stained with hematoxylin and eosin (H&E), Masson's Trichrome (Polysciences, Inc.) or using immunofluorescence techniques. Masson's Trichrome staining was carried out according to the manufacturer protocol with the addition of an one hour 10% formalin fix at room temperature (RT) prior to the fixation in Bouin's solution [50 ]. For immunostaining, the sections were fixed with 4% paraformaldehyde for 15 minutes at RT then blocked with 5% BSA/0.3% TritonX-100/PBS for one hour at RT, incubated with the primary antibody, 8-oxoG DNA Lesion (Santa Cruz), overnight at 4°C and incubated with secondary AlexFluor antibody (Invitrogen) at RT for 1 hour. Slides were mounted with Vectashield Hard Set with Dapi (Vector Laboratories). All images were taken with an Eclipse Ni microscope (Nikon).

Immunofluorescence experiments were performed according to [73 (link)]. Briefly, hippocampal primary cultures at DIV14 (see above) were fixed in 4% paraformaldehyde (PFA) and 4% sucrose solution for 30 min, followed by permeabilization with phosphate-buffered saline (PBS) at pH 7.4, containing 0.5% Triton X-100, for 3 min. Co-cultures were first blocked for 1 h in PBS containing 1% BSA, 0.2% Triton X-100, and subsequently incubated overnight at 4 °C with primary antibodies in PBS containing 1% BSA and 0.2% Triton X-100. The following antibodies were used: anti-GFP (AbCam Cambridge, UK Ab290), anti-PSD95 (AbCam Cambridge, UK Ab12093), anti-JIP1 (AbCam Cambridge, UK Ab24449), anti-β-arrestin2 (AbCam Cambridge, UK Ab31294), anti-JNK3 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA #PA5-14421). Cells were finally incubated with secondary antibodies (AlexFluor Antibody, Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. A concentration of 2 mg/mL Hoechst (Thermo Fisher Scientific, Waltham, MA, USA, 33342) was used to stain nuclei. ProLong Glass Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA) was used as a mounting agent.