LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute
LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute
LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute
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solution in TBS-T containing 0.02% sodium azide. The antibody dilutions of secondary<br />
goat anti-mouse HRP and goat anti-rabbit HRP antibodies were made in 2.5% milk<br />
(Carnation) in TBS-T containing 1% goat serum. Dilutions of the secondary anti-goat<br />
antibody were made in 2.5% milk (Carnation) in TBS-T without goat serum.<br />
3.19 Histology.<br />
For histological analysis of mutlitple tissues were fixed in 10% formaldehyde (SIGMA)<br />
overnight and processed for histology. Five micron sections of paraffin embedded tissue<br />
were prepared and haematoxylin/eosin staining was performed according to standard<br />
methods (197).<br />
3.20 Immunohistochemistry.<br />
For immunohistochemical staining of lung, spleen, testis and skin section, the mice were<br />
sacrificed and the tissues were fixed in 10% formaldehyde (SIGMA) overnight and<br />
processed for histology. 5µm sections of paraffin embedded tissue were prepared and<br />
permeabilized for antigen retrieval by microwaving the fixed tissue sections in 10mM<br />
Tris buffer (pH 9) with 2mM EDTA and immunohistochemical staining was performed<br />
using vectastain ABC kit according to manufacturer’s protocol.<br />
3.21 Immunofluorescence and confocal microscopy.<br />
5 m cryosections of multiple tissues were observed by confocal microscopy to detect the<br />
presence of EGFP-f. Confocal images were obtained by using a LSM 510 Meta Carl<br />
Zeiss Confocal system with an Argon 488 nm and Helium/Neon 543 nm lasers. All<br />
images were obtained using an Axio Observer Z.1 microscope (numerical aperture [NA]<br />
1.4) at a magnification of X 63. 5µm cryosections of the different tissues were<br />
deparafinzed and treated with 1% sodium borohydrate at room temperature to reduce<br />
auto-fluoresence of tissues. Blocking was performed with 1%BSA and tissues were<br />
further immunostained using GFP monoclonal antibody 1:50 (clonetech) overnight to<br />
detect the presence of EGFP-f. The next day, the sections were washed and incubated<br />
with the secondary antibody (Goat Anti mouse Alexa fluor 568 1:200 Invitrogen).<br />
Prpodium iodide was used to stain sperm DNA. Confocal images were obtained as<br />
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