Two types of immunofluorescence detections were performed on the BrdU-treated lizards. For those in the first group, with survival times of 1.5 h or 3 days (n = 3 each), a triple immunofluorescence detection for GFAP/DCX/BrdU was performed. For those in the second group, with a survival time of 7 days from the first injection and 3 days from the last injection (n = 3), a double immunofluorescence for BrdU/PCNA was performed.
In both cases the protocol for fluorescence immunohistochemistry was similar, except for the antibodies used. First, the slides were deparaffinized and hydrated. Then, they were treated with HCl 2N for 10 min at 37°C for DNA denaturation, rinsed in 0.1 M borate buffer and washed in phosphate buffered saline containing 0.1% Triton X-100 and BSA 0.1% (PTA). Subsequently, the sections were incubated in a blocking solution containing 10% casein (Vector) or 5% normal goat serum (NGS) (Sigma, San Luis, MO, USA) in PTA for 1 h, for triple or double immunoassay, respectively. After rinsing in PTA, the sections were incubated in blocking solution with the corresponding primary antibodies overnight at 4°C. The primary antibodies used for the first group were: mouse anti-BrdU (1:150, Dako), rabbit anti-GFAP (1:500, Dako), and goat anti-DCX (1:200, Sta. Cruz Biotechnologies); and for the second group: mouse anti-PCNA (1:500, Sigma, San Luis, MO, USA), and rat anti-BrdU (1:200, Abcam, Cambridge, UK). Sections were then washed with PTA and incubated with fluorescent secondary antibodies at 1:500 in blocking solution for 1 h at room temperature in the dark. The secondary antibodies used for the first group were: donkey anti-mouse Alexa 647 (1:500, Invitrogen, Walthan, MA, USA), donkey anti-rabbit Alexa 488 (1:500, Invitrogen, Walthan, MA, USA), and donkey anti-goat Alexa 555 (1:500, Invitrogen, Walthan, MA, USA); and for the second group: goat anti-mouse Alexa 555 (1:500, Invitrogen, Walthan, MA, USA), and goat anti-rat Alexa 488 (1:500, Invitrogen, Walthan, MA, USA). The sections were then washed in 0.1 M PB and incubated for 10 min with DAPI 1:1000 in H2O (Sigma, San Luis, MO, USA) at room temperature in the dark. Finally, the slides were washed with 0.1 M PB and mounted with Fluorsave (Calbiochem). The sections were analyzed with a Leica (Wetzlar, Germany) SP2 TCS AOBS inverted confocal microscope.
In both cases the protocol for fluorescence immunohistochemistry was similar, except for the antibodies used. First, the slides were deparaffinized and hydrated. Then, they were treated with HCl 2N for 10 min at 37°C for DNA denaturation, rinsed in 0.1 M borate buffer and washed in phosphate buffered saline containing 0.1% Triton X-100 and BSA 0.1% (PTA). Subsequently, the sections were incubated in a blocking solution containing 10% casein (Vector) or 5% normal goat serum (NGS) (Sigma, San Luis, MO, USA) in PTA for 1 h, for triple or double immunoassay, respectively. After rinsing in PTA, the sections were incubated in blocking solution with the corresponding primary antibodies overnight at 4°C. The primary antibodies used for the first group were: mouse anti-BrdU (1:150, Dako), rabbit anti-GFAP (1:500, Dako), and goat anti-DCX (1:200, Sta. Cruz Biotechnologies); and for the second group: mouse anti-PCNA (1:500, Sigma, San Luis, MO, USA), and rat anti-BrdU (1:200, Abcam, Cambridge, UK). Sections were then washed with PTA and incubated with fluorescent secondary antibodies at 1:500 in blocking solution for 1 h at room temperature in the dark. The secondary antibodies used for the first group were: donkey anti-mouse Alexa 647 (1:500, Invitrogen, Walthan, MA, USA), donkey anti-rabbit Alexa 488 (1:500, Invitrogen, Walthan, MA, USA), and donkey anti-goat Alexa 555 (1:500, Invitrogen, Walthan, MA, USA); and for the second group: goat anti-mouse Alexa 555 (1:500, Invitrogen, Walthan, MA, USA), and goat anti-rat Alexa 488 (1:500, Invitrogen, Walthan, MA, USA). The sections were then washed in 0.1 M PB and incubated for 10 min with DAPI 1:1000 in H2O (Sigma, San Luis, MO, USA) at room temperature in the dark. Finally, the slides were washed with 0.1 M PB and mounted with Fluorsave (Calbiochem). The sections were analyzed with a Leica (Wetzlar, Germany) SP2 TCS AOBS inverted confocal microscope.
Free full text: Click here
