Alexa fluor 488 donkey anti mouse

We conducted immunohistochemical analyses on the coronal brain slices to assess the expressions of glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 protein (Iba-1), and neuron-specific nuclear protein (NeuN) after blast exposure. The expressions of GFAP, Iba-1, and NeuN are associated with the distributions of astrocytes, microglia, and neuronal cells, respectively^{21 (link)}.
At FD Neurotechnologies, the brain slices were incubated with primary antibodies to detect GFAP (#556330; BD Biosciences, San Jose, CA; 1:350 dilution), Iba-1 (#019-19741; Wako Chemicals, Richmond, VA; 1:350 dilution), and NeuN (#ABN90P; Millipore Sigma, Burlington, MA; 1:250 dilution). This was followed by incubation in PBS containing the following secondary reporter antibodies with a fluorescent tag: Alexa Fluor 488 donkey anti-mouse (#A21202; Thermo Fisher Scientific, Waltham, MA; 1:250 dilution), Alexa Fluor 594 donkey anti-rabbit (#A21207; Thermo Fisher Scientific; 1:250 dilution), and Alexa Fluor 405 streptavidin conjugate (#S32351; Thermo Fisher Scientific; 1:200 dilution) for the detection of GFAP, Iba-1, and NeuN primary antibodies, respectively. The stained coronal slices were shipped back to their respective laboratories of origin (WRAIR or NJIT) for quantification analyses.

Antibody staining of cells was performed in the same way as described in siRNA Screening and Transfection. Plates were washed once with PBS and fixed with 4% paraformaldehyde incubated for 10 min at room temperature. Plates were then permeabilized by incubating with 0.2% Triton-X-100 in PBS for 5 min at room temperature, incubated with blocking buffer (10% FBS, 0.25% fish skin gelatin in PBS) for 1 h and stained overnight at +4 °C with the primary antibody anti-involucrin (SY3 or SY7 clones) or anti-cleaved caspase 3 (Asp175) (Cell Signaling catalog no. 9661) diluted to 1 µg/mL or according to manufacturer’s instructions in blocking buffer. Plates were then washed three times with PBS, stained with the secondary antibodies Alexa Fluor 555 donkey anti-mouse (Thermo Fisher Scientific) and/or Alexa Fluor 488 donkey anti-mouse (Thermo Fisher Scientific) at 1 µg/mL, the nuclear dye DRAQ5 (abcam) at 10 µM, and Alexa Fluor 647 Phalloidin (Thermo Fisher Scientific) at 12.6 nM in blocking buffer. Secondary stains were incubated for 2 h at room temperature protected from light, and plates were finally washed three times with PBS before being imaged using the Perkin-Elmer Operetta High-Content Imaging System.