Dako anti fade mounting medium

Immunofluorescence was used to double label astrocytes [mouse anti-glial fibrillary acidic protein (GFAP), 1:500, Sigma, cat#. G3893], and microglia [rabbit anti-ionized calcium binding adaptor molecule (Iba-1), 1:500, Wako, 019–19,741]. Brainstem sections were incubated at 60°C for 30 min and then dewaxed in xylene, rehydrated in ethanol, and washed in 0.1 mol/l phosphate buffer saline (PBS; pH 7.4). Heat mediated antigen retrieval was performed in citrate buffer (pH 6) using a microwave for 15 min. 10% normal goat serum (NGS) in 0.1% PBS + Triton X-100 (PBST) was used for non-specific antigen blocking. Sections were incubated in primary antibody and 0.1% PBST and 2% NGS overnight at 4°C. Negative controls omitting the primary antibody were included to confirm the absence of non-specific staining. Sections were incubated in 1:200 goat anti-mouse- Alexa Fluor 594 (Cat#: 115-585-003, Jackson ImmunoResearch, West Grove, PA, USA) and 1:200 goat-anti-rabbit- Alexa Fluor 488 (Cat#: 111-545-144, Jackson ImmunoResearch) in 0.1% PBST and 2% NGS for 2 h at room temperature. 1:1,000 HOECHST 33342 trihydrochloride, trihydrate (Cat#: H3570, Invitrogen, ThermoFisher Scientific) was used for nuclei staining, slides incubated for 5 min. Slides were washed in PBS and coverslipped using DAKO anti-fade mounting medium (Cat#: GM30411-2, Agilent technologies, CA, USA).

Cells were fixed with 4% (w/v) paraformaldehyde in PBS 24–48 h post-transfection. Specimens were permeabilized and blocked with 5% (w/v) BSA in PBS + 0.25% (v/v) Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) overnight at 4 °C, then incubated with primary antibodies diluted in 1% (w/v) BSA in PBS + 0.1% (v/v) Triton X-100. Cells were washed extensively, then diluted secondary antibodies were added and incubated for 2 h. 1 mM Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) was added for nuclear counterstaining. Specimens were mounted with Dako antifade mounting medium (Agilent, Santa Clara, CA, USA).