Alexa fluor 594 conjugated anti goat

Whole brain, nodose ganglion, and distal colon were immersed in 4% paraformaldehyde/phosphate‐buffered saline (PBS) for 24 h at 4°C, and then incubated for 24 h in phosphate buffer containing 20% sucrose, quickly frozen on dry ice, and cut into 8‐μm slices with a cryostat (Leica CM3050S; Leica) at −20°C. Sections were washed in PBS, blocked in Serum‐Free Protein Block (DAKO) for 10 min at room temperature, and then incubated overnight at 4°C with rabbit anti–ionized calcium binding adaptor molecule 1 (Iba1) (1:1000; WAKO), goat anti–Toll‐like receptor 4 (TLR4) (1:100; Santa Cruz Biotechnology), or rabbit anti‐mucin 2 (MUC2) (1:50; Santa Cruz Biotechnology). Sections were washed in PBS, and then incubated with Alexa Fluor 488–conjugated anti‐rabbit (1:1000; Invitrogen) or Alexa Fluor 594–conjugated anti‐goat (1:1000; Invitrogen) antibody for 1 h at room temperature. Nuclei were stained with DAPI. Images were captured on an Olympus AX‐7 fluorescence microscope or a Confocal Microscope C2 (Nikon). TLR4 and MUC2 pixel intensities were determined automatically using the cellSens Dimension imaging software (Olympus). Iba1‐positive cells were counted manually using the same software. Quantitation was performed in a blinded fashion.

Details of the plasmids cloned for the study are available on request.
Plasmids that have been previously described include: NLS-MKL1 (ref. 5 (link)); MKL1(2-263)-2GFP-N3 (RPEL), MC-pEGFP-C1 and pET41a-3CΔ-MKL1(2-261)11 (link); MKL1(fl)-C471 (ref. 7 (link)); and SRF-VP16 (ref. 36 (link)). RanBPI-ΔNES-pEGFP-C1 and Smad4-pEGFP-C1 were kind gifts from Caroline Hill, pQE-RanQ69L from Maarten Fornerod and pRL-TK was from Promega.
The following antibodies were from Sigma: Flag (M2, F1804, 1:300), haemagglutinin (HA-7, G21234, 1:300), Ipoβ (31H4, I2534, 1:2,000) and Tubulin (B512, T6074, 1:2,000). MKL1 (C-19, SC-21558, 1:100) antibody was from Santa Cruz Biotechnology and Ddx19 (NB100-760, 1:2,000) was from Novus Biologicals. The following secondary antibodies were from Invitrogen: horseradish peroxidase (HRP)-conjugated anti-mouse (G21040, 1:5,000), HRP-conjugated anti-rabbit (G21234, 1:5,000), HRP-conjugated anti-goat (A9452, 1:5,000), Alexa-Fluor-488-conjugated anti-mouse (A21155, 1:500), Alexa-Fluor-488-conjugated anti-rabbit (A11008, 1:500), Alexa-Fluor-594-conjugated anti-mouse (A11005, 1:500), Alexa-Fluor-594-conjugated anti-rabbit (A11012, 1:500) and Alexa-Fluor-594-conjugated anti-goat (A11080, 1:500).