The 732 bp coding sequence for zebrafish kinesin-12 (GeneBank XM_002666923) was amplified by PCR using the following primers: Left primer, 5′-tgtgctgctggagttaatgc-3′; Right primer, 5′-ttttgtgcgttgcttttctg-3′. Digoxigenin (DIG)-labeled RNA sense and antisense probes were made from the linearized plasmids according to the manufacturer’s protocol using the DIG RNA Labeling Kit (SP6/T7) (Roche). The procedure for in situ hybridization followed our protocol (Huang, Wang et al. 2013 (link)) which was modified from a previous study (Thisse and Thisse 2008 ). The small baskets were not used in our protocol. BM purple AP substrate (Roche) was used instead of the staining solution. We use the BBR (Boehringer blocking reagent, Roche) for blocking.
Boehringer blocking reagent
Manufactured by Roche
Sourced in United States
Boehringer Blocking Reagent is a laboratory product designed to block non-specific binding in various immunoassay and immunohistochemistry applications. It is a ready-to-use solution that helps to reduce background signal and improve the specificity of target detection.
Automatically generated - may contain errors
Lab products found in correlation
Bm purple ap substrate, by Roche (2 mentions)
Dig rna labeling kit sp6 t7, by Roche (1 mentions)
Sheep serum, by Merck Group (1 mentions)
Anti digoxigenin antibody, by Roche (1 mentions)
Bm purple, by Roche (1 mentions)
Stemi sv11 stereomicroscope, by Zeiss (1 mentions)
Dc 300 fx, by Leica camera (1 mentions)
Dm 5000b fluorescence microscope, by Leica camera (1 mentions)
Thermobrite system, by Abbott (1 mentions)
Pepsin, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Vector Laboratories (1 mentions)
Nitro blue tetrazolium chloride, by Roche (1 mentions)
G 50 micro columns, by GE Healthcare (1 mentions)
T7 rna polymerase, by Promega (1 mentions)
Dntps, by Roche (1 mentions)
5 bromo 4 chloro 3 indolyl phosphate, by Roche (1 mentions)
T3 rna polymerase, by Roche (1 mentions)
Ap conjugated anti digoxigenin antibody, by Roche (1 mentions)
Dig rna labeling kit, by Roche (1 mentions)
6 protocols using boehringer blocking reagent
1
Zebrafish Kinesin-12 mRNA Localization
2
In Situ Hybridization Protocol for Gad1 Transcript Detection
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Gad1 antisense RNA probe was transcribed in vitro from full-length cDNA template (IMAGE ID: 5358787). The probe was diluted to a final concentration of 800 ng/ml in hybridisation buffer (50% formamide, 10% dextran sulphate, 1 mg/ml rRNA, 1× Denhardt’s solution, 0.2 M NaCl, 10 mM Tris HCl, 5 mM NaH2PO4.2H2O, 1 mM Tris base, 50 mM EDTA) and applied onto the slides, which were incubated in a humidified chamber at 65°C overnight. The slides were then washed three times for 30 min in wash buffer (50% formamide, 1× SSC, 0.1% Tween) at 65°C, two times for 30 min in MABT buffer (100 mM maleic acid, 150 mM NaCl, 0.1% Tween-20) at RT, and blocked for 2 hr at RT (2% Boehringer Blocking Reagent (Roche), 20% inactivated sheep serum in MABT). Sheep a-DIG alkaline phosphatase conjugated antibody (Roche, 11093274910) was diluted 1:2000 in the blocking solution and incubated with the slides overnight at 4°C. This was followed by five 20 min washes in MABT and two 20 min washes in the AP buffer (0.1M Tris-HCl pH 8.2, 0.1%-Tween-20). Fast red TR/Naphthol AS-MX tablets (Sigma) were dissolved in the AP buffer and applied onto the slides for colour reaction for 3–6 hr at RT in the dark. The slides were then washed three times for 20 min in PBS before proceeding with IHC for GFP as described above. Sox14GFP/+ and Sox14GFP/GFP sections were always processed in parallel.
3
In Situ Hybridization Protocol for Embryos
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Fixed HH24, HH26 and HH28 embryos (n = 8 per treatment group) were dehydrated in increasing concentrations of methanol (MeOH). The processed embryos were stored at −20 °C for a maximum period of 2 weeks. Embryos were hydrated in decreasing concentrations of methanol. The embryos treated with Proteinase K, refixed in 4% PFA and treated with 75% post‐hybridisation buffer followed by pre‐hybridisation buffer. The ISH probes WT1 and TCF21 were added to the Prehybridization solution in a concentration of 530 ng μl−1 and left for 24 h at 65 °C.
After hybridisation, the embryos were washed with decreasing saline‐sodium citrate (SSC), followed by RNase treatment and blocking by 2% Boehringer blocking reagent (Roche) with 20% sheep serum (Sigma). Finally, embryos were incubated with anti‐digoxigenin antibody (1 : 5000; Roche) and colour development was carried out with 50% BM purple (Roche). The embryos were photographed in 100% glycerol using a Stemi SV 11 stereomicroscope (Carl Zeiss).
After hybridisation, the embryos were washed with decreasing saline‐sodium citrate (SSC), followed by RNase treatment and blocking by 2% Boehringer blocking reagent (Roche) with 20% sheep serum (Sigma). Finally, embryos were incubated with anti‐digoxigenin antibody (1 : 5000; Roche) and colour development was carried out with 50% BM purple (Roche). The embryos were photographed in 100% glycerol using a Stemi SV 11 stereomicroscope (Carl Zeiss).
4
MCPyV DNA Detection by FISH
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MCPyV FISH was performed as previously described.22 , 23 In brief, deparaffinized 3 μm thick sections were pretreated with 0.2 M hydrochloric acid, incubated with 1 M NaSCN and digested with 1 mg/mL pepsin (2500–3500 U/mg, Sigma Chemical, St. Louis, MO, USA). The biotin labeled “specific” MCPyV DNA probe was added to the samples at a concentration of 5 ng/μL, followed by denaturation of DNA (five minutes, 80°C) and hybridization overnight (37°C, humid chamber; ThermoBrite System, Abbott Molecular, Abbot Park, IL, USA). Unbound MCPyV DNA probe was stringently washed away. Bound probe was detected by sequential incubation in a combination of secondary antibodies: fluorescein isothiocyanate (FITC) avidin secondary antibody (1:500) and biotin conjugated goat anti‐avidin (1:100; Vector, Brunschwig Chemie, Amsterdam, The Netherlands). Prior to incubation, aspecific binding sites were blocked with Boehringer Blocking reagent (Roche,
Molecular Diagnostics Inc., South Branchburg, NJ, USA). Cell nuclei were counterstained, and cover slipped with 4,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI; 0.2 μg/mL; Vectashield, Vector Laboratories, Burlingame, CA, USA). Samples were visualized using a DM 5000B fluorescence microscope (Leica, Wetzlar, Germany) coupled to an online digital camera (Leica DC 300 Fx) for independent evaluation of FISH signals by two investigators.
Molecular Diagnostics Inc., South Branchburg, NJ, USA). Cell nuclei were counterstained, and cover slipped with 4,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI; 0.2 μg/mL; Vectashield, Vector Laboratories, Burlingame, CA, USA). Samples were visualized using a DM 5000B fluorescence microscope (Leica, Wetzlar, Germany) coupled to an online digital camera (Leica DC 300 Fx) for independent evaluation of FISH signals by two investigators.
5
Procedure for lft2 antisense probe synthesis
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The template for lft2 antisense probe was generated by PCR amplification of cDNA from 12 hours post fertilization wild-type zebrafish, using the following primers: SP6-lft2-F 5′-GATTTAGGTGACACTATAGgaccacagcgatctcactca-3′ and T7-lft2-R 5′-TAATACGACTCACTATAGGGgactggagggattttgtcc-3′. The PCR product was gel extracted and purified by ethanol precipitation. 1.5 μg of template DNA was transcribed using T7 RNA polymerase (Promega, #P207B) in the presence of digoxygenin (DIG)-labelled dNTPs (Roche, #12430721). Probes were purified using G-50 micro-columns (GE Healthcare, #28-9034-08). Embryos were permeabilized with 1% H2O2, equilibrated with hybridization buffer (50% Formamide, 1.3 × SSC pH 5.0, 5 mM EDTA pH 8.0, 200 μg ml−1 Baker’s yeast tRNA, 0.2% Tween-20, 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 100 μg ml−1 Heparin) and incubated overnight with lft2 DIG-labelled probes. Embryos were blocked with 20% sheep serum+2% Boehringer Blocking Reagent (Roche, #11096176001). Probes were detected with an anti-DIG-alkaline phosphatase antibody (Roche, #11093274910) and the signal was developed using 30 μg ml−1 of each nitro-blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate (Roche, #11383213001/#11383221001).
6
Whole-Mount In Situ Hybridization Protocol
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Embryos were fixed overnight at 4 °C in 4% paraformaldehyde (PFA) in PBS, dehydrated in increasing methanol concentrations in PBST (PBS + 0.1% Tween-20), and stored in 100% methanol at −20 °C until further processing, according to a standard protocol with minor modifications. Briefly, to detect the hybridized signal, embryos were washed with MBST (100 mM maleic acid, 150 mM NaCl, containing 0.1% Tween-20), pre-blocked in 10% sheep serum, 2% Boehringer Blocking Reagent (Roche) at room temperature for 90 min and then incubated overnight at 4 °C with a 1:2000 dilution of an alkaline phosphatase (AP)-conjugated anti-digoxigenin antibody (Roche) in blocking solution. After several washes in MBST of 1 h each, an additional overnight wash at 4 °C was performed. Detection of staining was performed using the BM purple AP substrate (Roche) for 8 h. The embryos were post-fixed with 4% PFA overnight at 4 °C and stored in 70% ethanol. Sense (negative-control) and antisense RNA probes for Strap were generated by PCR. Digoxigenin labeling was performed using the DIG RNA Labeling kit and T3 RNA polymerase (Roche). Primer sequences are listed in Supplementary Data 10 .
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