5 bromo 4 chloro 3 indolyl phosphate bcip

In situ hybridization studies were performed as previously described (Manieri et al., 2012 (link)). Briefly, digoxigenin-labeled antisense RNA probes were synthesized from a cDNA clone for Ptgs2 (clone ID: 30059181, Thermo Fisher Scientific) with T7 RNA polymerase (New England Biolabs, Ipswich, MA) and DIG RNA Labeling Mix (Roche, Indianapolis, IN) and purified with NucAway Spin Columns (Life Technologies, Carlsbad, CA). Deparaffinized and protease K-treated sections were hybridized with RNA probes and incubated with anti-digoxigenin alkaline phosphatase (AP)-conjugated antigen binding fragments (Fab) (Roche). Specific signals were visualized with nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Roche) and sections were counterstained with methyl green.

Purified proteins were separated on 4–20% gradient Tris–glycine SDS-PAGE gels and stained with Coomassie Blue R-250. For western blotting, proteins were transferred to 0.2 µm nitrocellulose membranes (Invitrogen, Waltham, MA) and blocked in PBS (pH 7.4), 0.1% Tween 20 (PBS-T) with 0.5% non-fat dry milk for 30 min at room temperature (RT). Western blots were probed with PfCelTOS-specific mouse mAbs 3D11.D4, 4H9.C3 and 3C3.B3 and anti-Histidine mouse mAb (at 1:3000 each) (Takara Bio, Mountain View, CA) for 1 h. After washing with PBS-T, blots were probed with alkaline phosphatase (AP)-conjugated anti-mouse IgG (1:5000) (Southern Biotech, Birmingham, AL). Blots were washed with PBS-T and developed for 10 min at RT with 4-nitro-blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Roche, Branchburg, NJ) in 0.1 M sodium chloride, 0.005 M magnesium chloride and 0.1 M Tris–HCl pH 9.

Throughout this work, the day of finding a copulation plug was counted as embryonic day (E)0.5, and the day when mice were born was counted as postnatal day (P)0. AP staining was performed on 200-μm-thick brain sections from P36 mice. Mice were anesthetized with ketamine/xylazine and then sacrificed by trans-cardiac perfusion with 4 % paraformaldehyde (PFA) [w/v, dissolved in phosphate buffered saline (PBS), pH 7.4], and brains were dissected out and further fixed in 4 % PFA overnight at 4 °C. Brains were embedded in 3 % low melting point agarose (w/v, dissolved in PBS) and sectioned at 200 μm on a vibratome. AP histochemistry was performed essentially as previously described [17 (link), 18 (link), 71 (link)]. Vibratome sections in PBS containing 2 mM MgCl₂ were heated in a water bath at 69 °C for 90 min and then equilibrated in AP-staining buffer (0.1 M Tris, 0.1 M NaCl, 50 mM MgCl₂, pH 9.5) overnight at room temperature. AP histochemistry was carried out in AP-staining buffer containing 0.34 μg/ml 4-nitro blue tetrazolium chloride (NBT) and 0.175 μg/ml 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) (Roche Applied Science; Indianapolis, IN) for 8 h at room temperature with gentle horizontal shaking. Sections were dehydrated through an ethanol series and cleared with BBBA [2:1, benzyl benzoate (BB)/benzyl alcohol (BA)] (Sigma-Aldrich; St. Louis, MO) before imaging.

We generated an anti-sense probe with 3′ ~500 bp mouse syntaxin-3 cDNA which was subcloned in PstI-EcoRI site of pBluescript SKII. The plasmids were then linearized by digesting with BamHI and subjected to in vitro transcription. In situ hybridization on the coronal hippocampus section was carried out as described previously^{15 (link)}. Briefly, digoxigenin (DIG) labeled anti-sense RNA probe for syntaxin-3 were generated according to manufacture’s protocol (Roche, Laval, Canada). Alkaline phosphatase (AP)-conjugated anti-DIG antibody (Roche) was used to detect hybridized probe. AP activity was detected by 5-bromo-4-chloro- 3-indolyl-phosphate (BCIP, Roche) and 4-nitoro blue tetrazolium chloride (NBT, Roche) in NTMT (100 mM NaCl, 100 mM Tris-Cl pH9.5, 50 mM MgCl2, 0.1% Tween20, 2 mM levamisol).

Interested cells were lysed in 1× loading buffer (80 mM Tris, pH 6.8, with 2.0% sodium dodecyl sulfate [SDS], 10% glycerol, 100 mM dithiothreitol, and 0.2% bromophenol blue). The samples were boiled for 30 min, and proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were transferred onto nitrocellulose membranes (GE Whatman, UK) and probed with various primary antibodies against the indicated proteins (see the figure legends). Secondary antibodies were alkaline phosphatase-conjugated anti-rabbit (Jackson, USA) and anti-mouse (Jackson, USA), and staining was carried out with 5-bromo-4-chloro-3-indolylphosphate (BCIP) (Roche, Switzerland) and nitroblue tetrazolium (NBT) (Sigma-Aldrich, USA). For each lane of protein gels, 30 μg total protein was loaded for the detection of exogenous proteins and loading controls, while 50 μg was loaded for the detection of endogenous proteins.

Digoxigenin (DIG)-labeled Foxg1 antisense probes were prepared as previously described^{61 (link)}. Egr1 and Egr2 antisense probes were prepared from FANTOM clones (I730052D02 for Egr1 and E430002H10 for Egr2) and used as previously described^{62 (link)}. Briefly, samples were pretreated and hybridized with probes (100 ng/ml) at 55 °C overnight. The anti-DIG antibody (1:500, Roche) was applied at 4 °C overnight. Foxg1 signals were developed using Texas-Red streptavidin (1:500, Vector) at room temperature for 2 h. After the tissue was washed with PBS, immunostaining was performed from a blocking step with 10% normal donkey serum and 0.05% Triton X-100 in PBS. The primary antibody, mouse anti-COUP-TFI (1:200, Perseus Proteomics), was diluted in 1% normal donkey serum and 0.05% Triton X-100 in PBS and incubated at 4 °C overnight, and an Alexa Fluor 488-conjugated secondary antibody was used. For Egr1 and Egr2 probes, signals were developed with nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) substrates (Roche) at room temperature. Images were acquired using an Axio Imager Z1 fluorescence microscope (Carl Zeiss).