Mice were euthanized at postnatal 30 days, and tracheal blocks and other organs were dissected, formalin-fixed and embedded in paraffin and 5-µm sections were prepared for subsequent analyses. To determine parathyroid gland size, appropriate tissue sections were digitalized and maximal parathyroid cross-sectional area was measured by computerized pixel counting. For immunofluorescence (IF) staining, paraffin sections were incubated with primary antibodies to calcium-sensing receptor (CASR) (1:50, rabbit polyclonal antiserum #33821, Santa Cruz Biotechnology), PTH (1:200, mouse monoclonal antiserum AB63993, Abcam), GCM1 (1:50, rabbit polyclonal antiserum 98811, Santa Cruz Biotechnology) or GCM2 (1:50, goat polyclonal antiserum 79495, Santa Cruz Biotechnology) at 4° C overnight. Antibody binding was detected by incubation with appropriate secondary antibodies (anti-mouse Alexa Fluor 647, anti-rabbit Alexa Fluor 488, and anti-goat Alexa Fluor 488, Invitrogen) at 1:200 dilution for 45 minutes in the dark. Finally, the sections were stained with 4′6-diamidino-2-phenylinodole (DAPI) (Vector Laboratories, Burlingame, CA) and imaged by fluorescence microscopy.
4 6 diamidino 2 phenylinodole dapi
Manufactured by Vector Laboratories
DAPI is a fluorescent dye that binds strongly to the minor groove of DNA. It is commonly used in various biological applications, such as cell staining and fluorescence microscopy, to visualize and identify cell nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexafluor 647 conjugated donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Cy3 conjugated donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Axiovert 200, by Zeiss (1 mentions)
Alexa fluor 488 conjugated donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Rabbit monoclonal anti vimentin antibody, by Abcam (1 mentions)
3 protocols using 4 6 diamidino 2 phenylinodole dapi
1
Parathyroid Gland Morphometry and Immunohistochemistry
2
Immunohistochemical Analysis of Transplanted Bone Marrow
3
Cellular Imaging of MMP13 Inhibitors
Check if the same lab product or an alternative is used in the 5 most similar protocols
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SFs and
HUVECs were cultured as described above. First, 5 × 103 cells were seeded per well in Nunc Lab-TekII-CC2 8-chambered slides.
After 24 h, the medium was changed and to it was added PPMMP13B, PPMMP13, or DPPMMP13B to a final concentration
of 2.5 μM. The cells were incubated for an additional 24 h.
Wells were washed with PBS, and cells were subsequently fixed with
500 μL of 4% paraformaldehyde in PBS for 20 min, after which
the fixed cells were washed with PBS twice. The chamber was then removed,
and 10 μL of mounting solution with 4′,6-diamidino-2-phenylinodole
(DAPI) (Vector Laboratories Inc.) was added to each well. The cells
were covered by a cover slide and subjected to microscopic studies
using an Olympus 1X73 inverted microscope (pyro: Cy5 620/60 nm excitation,
665 nm long-pass emission, nuclei: DAPI 375/28 nm excitation, 460/50
nm emission).
HUVECs were cultured as described above. First, 5 × 103 cells were seeded per well in Nunc Lab-TekII-CC2 8-chambered slides.
After 24 h, the medium was changed and to it was added PPMMP13B, PPMMP13, or DPPMMP13B to a final concentration
of 2.5 μM. The cells were incubated for an additional 24 h.
Wells were washed with PBS, and cells were subsequently fixed with
500 μL of 4% paraformaldehyde in PBS for 20 min, after which
the fixed cells were washed with PBS twice. The chamber was then removed,
and 10 μL of mounting solution with 4′,6-diamidino-2-phenylinodole
(DAPI) (Vector Laboratories Inc.) was added to each well. The cells
were covered by a cover slide and subjected to microscopic studies
using an Olympus 1X73 inverted microscope (pyro: Cy5 620/60 nm excitation,
665 nm long-pass emission, nuclei: DAPI 375/28 nm excitation, 460/50
nm emission).
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