Multi-color FISH was performed as previously described in (35 (link)) with minor modifications. Tissues were serially rehydrated, and the pituitary was detached from the brain. Afterwards, whole pituitaries were hybridized with the probes (0.11 – 3.17 ng/µl) for 18 hours at 55°C, and incubated with different combinations of anti-DNP- (Perkin Elmer), anti-FITC-, and anti-DIG-conjugated antibodies (Roche Diagnostics), followed by TAMRA- (Thermofisher), Cy5- (Perkin Elmer) and FITC-conjugated tyramides (Sigma). The nuclei were stained with DAPI (1:1000, 4’, 6-diamidino-2-phenylindole dihydrochloride; Sigma). The absence of labeling when using sense probes was used to confirm the specificity of the anti-sense probes. Whole pituitaries were mounted using Vectashield H-1000 Mounting Medium (Vector, Eurobio/Abcys) between microscope slides and cover slips (Menzel Glässer, VWR) with spacers (Reinforcement rings, Herma) in between for the juveniles, and between two cover slips with spacers for adults.
Fitc conjugated tyramides
Manufactured by Merck Group
FITC-conjugated tyramides are a type of laboratory reagent used in various biological and immunological assays. They are composed of fluorescein isothiocyanate (FITC) molecules conjugated to tyramide, a small organic compound. FITC-conjugated tyramides are employed as signal amplification reagents, enhancing the detection of target analytes in techniques such as immunohistochemistry and in situ hybridization.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield mounting medium h 1000, by Vector Laboratories (2 mentions)
Tamra, by Thermo Fisher Scientific (2 mentions)
Anti dnp, by PerkinElmer (2 mentions)
Anti dig conjugated antibodies, by Roche (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride, by Merck Group (1 mentions)
Vibratome, by Leica (1 mentions)
4 6 diamidino 2 ph nylindole dapi, by Merck Group (1 mentions)
H 1000 mounting medium, by Vector Laboratories (1 mentions)
3 protocols using fitc conjugated tyramides
1
Multi-color FISH Assay for Pituitary Cells
2
In Situ Hybridization of Mouse Brain-Pituitary Complex
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FISH was performed as previously described (13 , 33 (link)) with some minor adjustments. After rehydration in a series of decreasing ethanol concentrations, the brain–pituitary complex was hybridized with previously validated specific probes (0.11-3.17 ng/µL) (Table 2 ) for 18 hours at 55 °C and incubated with different combinations of antifluorescein isothiocyanate (FITC)- and antidigoxigenin-conjugated antibodies (Roche Diagnostics), followed by tetramethylrhodamine- (Thermofisher) or FITC-conjugated tyramides (Sigma). After staining nuclei with 4',6-diamidino-2-phénylindole (DAPI; 1:1000; Sigma) and several washes with PBST, the tissue was embedded in 3% agarose (in H2O) and sectioned parasagitally at a thickness of 70 µm using a vibratome (Leica). The slices were mounted using Vectashield H-1000 Mounting Medium (Vector, Eurobio/Abcys) between microscope slides and coverslips (Menzel Glässer, VWR). For whole pituitary mounting, spacers (Reinforcement rings, Herma) were added in between.
3
Multi-color FISH Pituitary Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Multi-color FISH was performed as previously described in (34) with minor modifications. Tissues were serially rehydrated, and the pituitary was detached from the brain. Afterwards, whole pituitaries were hybridized with the probes (0.11 -3.17 ng/µl) for 18 hours at 55 o C, and incubated with different combinations of anti-DNP-(Perkin Elmer), anti-FITC-, and anti-DIG-conjugated antibodies (Roche Diagnostics), followed by TAMRA-(Thermofisher), Cy5-(Perkin Elmer) and FITC-conjugated tyramides (Sigma). The nuclei were stained with DAPI (1:1000, 4', 6diamidino-2-phenylindole dihydrochloride; Sigma). The absence of labeling when using sense probes was used to confirm the specificity of the anti-sense probes. Whole pituitaries were mounted using Vectashield H-1000 Mounting Medium (Vector, Eurobio/Abcys) between microscope slides and cover slips (Menzel Glässer, VWR) with spacers (Reinforcement rings, Herma) in between for the juveniles, and between two cover slips with spacers for adults.
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