A6397

The last sections through the Vc of the rats in the first group and the sections through the Vc from the second group injected with FG, TMR, and formalin were also processed for triple-immunofluorescence histochemistry for Fos, TMR and FG. Briefly, the sections were incubated with (1) a mixture of 1 μg/ml mouse anti-Fos antibody (ab 208,942, Abcam, Cambridge, MA, USA), 1 μg/ml guinea pig anti-FG antibody (NM-101, Protos Biotech Corporation) and 0.5 μg/ml rabbit anti-TMR antibody (A-6397, Invitrogen) in PBS that contained 0.3% (v/v) Triton X-100, 0.25% (w/v) λ-carrageenan, and 3% (v/v) donkey serum (PBS-XCD) overnight at room temperature; (2) 10 μg/ml biotinylated donkey anti-mouse IgG (AP192B, Millipore, Temecula, CA, USA); and (3) a mixture of 10 μg/ml Alexa 647-conjugated goat anti-guinea pig IgG antibody (A-21450, Invitrogen), 10 μg/ml Alexa594-conjugated donkey anti-rabbit IgG antibody (A-21207, Invitrogen), and 10 μg/ml Fluorescein Avidin D (A-2001; Vector) in PBS that contained 3% (v/v) normal donkey serum.
Another series of sections from the second group was used to conduct the control experiments for immunofluorescence histochemistry, in which the Fos antibody was omitted. Under these conditions, no immunoreactivity for the omitted antibody was observed.

Imaging flow chambers were constructed as in Section VII of Gell et al., 2010 (link), with the following modifications: two narrow strips of parafilm replaced double-sided scotch tape as chamber dividers: following placement of the smaller coverslip onto the parafilm strips, the chamber was heated to melt the parafilm and create a seal between the coverslips; typically, only three strips of parafilm were used, resulting in two chambers per holder. Chambers were prepared with an anti-rhodamine antibody (Invitrogen A6397, RRID:AB_2536196) followed by blocking with Pluronic F127, as described in Section VIII of Gell et al., 2010 (link). Microtubules were adhered to the chamber coverslip, and the chamber was flushed gently with warm BRB80. The flow chamber was heated to 28 °C using an objective heater on the microscope stage, and then 3–4 channel volumes of imaging buffer were flushed through the chamber. Microtubules were imaged on a Nikon TiE microscope using 488 nm and 561 nm lasers sent through a Ti-TIRF-PAU for Total Internal Reflectance Fluorescence (TIRF) illumination. An Andor iXon3 EM-CCD camera fitted with or without a 2.5x projection lens depending on the experiment was used to capture images with high signal-to-noise and small pixel size (64 nm or 160 nm, respectively). Images were collected using TIRF with a Nikon CFI Apochromat 100x 1.49 NA oil objective.

Cells were incubated with culture media containing the YnMyr (20 µM, WuXi AppTec, Shanghai, China) for 24 h before harvest. Then, a click mixture [mixed with the capture reagent AzTB (0.1 mM, WuXi AppTec), CuSO₄ (1 mM), tris(2-carboxyethyl)phosphine (TCEP) (1 mM), and tris(benzyltriazolylmethyl)amine (TBTA) (0.1 mM) in DMSO] was added to each sample before vortexing at RT for 1 h. After adding 1 ml ice-cold MeOH containing EDTA (10 mM), the samples were quickly vortexed and kept at −80°C overnight. Next day, the samples were centrifuged to pellet precipitated proteins, and the pellets were dissolved by 75 µl 2% SDS in PBS and 25 µl 4 × SLB [sample-loading buffer prepared by mixing 4 × NuPAGE™ LDS sample buffer (Thermo Fisher Scientific, Inc. Wal-tham, MA, USA) and ß-mercaptoethanol of a ratio of 5:1]. Finally, the samples were subjected to WB using primary antibody against TAMRA (Abcam, #ab171120 or Invitrogen, #A6397).