Anti dig alkaline phosphatase fab fragments

As previously described (Pusapati et al., 2018 (link)), to generate a Mosmo in situ probe, Mosmo specific primers were designed using the program Primer3: forward 5′-acacgtgtgtgctgaaaagc-3′ and reverse 5′-gagattaaccctcactaaagggatgagcaggtaacccatctcc-3′. The underlined sequence marks the T3 polymerase binding site that was incorporated into the reverse primer. The Mosmo probe was generated using a digoxigenin (DIG) RNA Labeling Kit (Roche). Briefly, the probe was generated from the in vitro transcription of PCR products amplified from mouse neural progenitor cell cDNA. After overnight hybridization at 65°C, the signal was visualized using Anti-DIG-alkaline phosphatase (AP) Fab fragments (Roche) and NBT/BCIP (Roche).

RNA in situ hybridization on the mouse brain sections was performed with digoxigenin (DIG)-labeled RNA probes. Full-length cDNA of Chd8 was amplified with specific PCR primers and cloned into a pGEM-T easy vector (Promega) to generate an antisense probe for Chd8. The DIG-labeled antisense probes were synthesized by in vitro transcription using SP6 RNA polymerase. Mice of different developmental stages were perfused with 4% PFA, and fixed brains were sectioned into 20-μm slices using a cryostat. RNA in situ hybridization was performed as described previously [26 (link)]. Brain sections were hybridized for 18 h at 60 °C. The hybridization signal was detected with anti-DIG–alkaline phosphatase Fab fragments (Roche) and nitro blue tetrazolium chloride (NBT) plus 5-bromo-4-chlor-indolyl-phosphate (BCIP) as color reaction substrates.

To confirm the in situ hybridization specificity, two different single stranded digoxigenin (DIG)-labeled RNA probes of 476 bp and 604 bp lengths corresponding to different regions of LsMTP transcripts (Fig. 1) were synthesized separately from cDNA using the DIG RNA labeling kit (Roche). Primers used for the synthesis of sense and antisense RNA probes are listed in supplemental Table S1. The concentration and labeling efficiency of probes was assessed by spectrometry (Nanodrop ND-1000) and with a spot test on nylon membrane, respectively. In situ hybridization was carried out in paraffin-embedded sections of adult female lice, as previously described by Kvamme, Frost, and Nilsen (24 (link)) and Dalvin, Nilsen, and Skern-Mauritzen (25 ) with some modifications. Tissue sections were deparaffinized with Histoclear (National Diagnostic) instead of xylene and proteinase K treatment was done for 13 min. Hybridization of probes (500 ng/100 μl) was carried out at 65°C for 16–20 h. Sections were incubated with anti-DIG-alkaline phosphatase Fab fragments (Roche) and visualized using nitroblue tetrazolium (Roche) and 5-bromo-4-chloro-3-indolyl phosphate (Roche). The localization of LsMTP transcripts was detected with antisense probes and sense probes were used as negative controls.

The cDNAs used as templates to make probes were prepared by RNA extraction and reverse transcription PCR (RT-PCR). The primers for RT-PCR were designed according to the Esembl genomic sequences [19 (link)]. In situ hybridization was performed as described previously [20 (link)] with minor modifications. Briefly, DIG-labeled riboprobes were incubated with the embryos to detect the expression pattern of CRMP2 and CRMP4. Anti-DIG-alkaline phosphatase Fab fragments (Roche, Mannheim, Germany) and NBT/BCIP (Sangon Biotech, Shanghai, China) were used to detect and amplify the signals.

Single stranded Digoxigenin (DIG) labelled RNA probe of 571 nt was synthesized from cDNA using the DIG RNA labelling kit (Roche). Primers used for the synthesis of sense and antisense RNA probes are listed in Table 1. The concentration and quality of the probes were determined by spot test and spectrometry (Nanodrop ND-1000). In situ hybridization was performed in paraffin embedded sections of adult female lice and copepodids as previously described by Dalvin et al. [42 ] and Eichner et al. [43 (link)] with some modifications. Histoclear (National Diagnostic) was used to deparaffinize the sections and proteinase K treatment was carried out for 18 minutes. Sections were hybridized with DIG-labeled RNA probes (1500 ng/100μl) at 65°C for 20 hr. Afterward, sections were incubated with anti-DIG-alkaline phosphatase Fab fragments (Roche) and visualized using the nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Roche). Sense probe was used as a negative control. Pictures were obtained with an Axio Scope.A1 microscope (Zeiss).