Monoclonal anti βiii tubulin antibody

Fixed cells were washed in several changes of phosphate buffered saline (PBS) before permeablisation and blocking in PBS containing 3% bovine serum albumin (BSA) and 0.1% Triton X-100 (both from Sigma, Poole, UK). Cells were then incubated with monoclonal anti-βIII-tubulin antibody (Cat No. T8660; 1:200 dilution; Sigma) for 1 h at room temperature (RT) and used to localise RGC and their neurites. Cells were then washed in several changes of PBS and incubated with Alexa 488 anti-mouse IgG (Cat No. A-11001; 1:400 dilution; Invitrogen) for 1 h at RT, washed in several changes of PBS and mounted using Vectashield containing DAPI (Vector Laboratories, Peterborough, UK). Slides were then anonymised by a second investigator and viewed using a Zeiss epi-fluorescent microscope equipped with an AxioCam HRc and Axiovision Image capture software (all from Zeiss, Hertfordshire, UK). Immunocytochemistry included controls with primary antibody omitted, that were used to set background levels of nonspecific staining (not shown) prior to image capture.

Fixed cells were washed in several changes of PBS before permeabilisation and blocking of non-specific antibody sites with PBS containing 3% bovine serum albumin (BSA) and 0.1% Triton X-100 (both from Sigma, Poole, UK). Cells were then incubated with monoclonal anti-βIII-tubulin antibody (1:200 dilution; Sigma) for 1 h at room temperature (RT) to detect retinal neurons and their neurites. Cells were then washed in several changes of PBS and incubated with Alexa 488 anti-mouse IgG (1:400 dilution; Invitrogen) for 1 h at RT. After washing in several changes of PBS and mounting in Vectamount containing DAPI (Vector Laboratories, Peterborough, UK), cells were then viewed using a Zeiss epi-fluorescent microscope equipped with an AxioCam HRc and Axiovision Image capture software (all from Zeiss, Hertfordshire, UK). Immunocytochemistry included controls with primary antibody omitted, to set background levels of nonspecific staining (not shown) before image capture.

DRGN were fixed in 4% (v/v) paraformaldehyde (TAAB Laboratories, Berkshire, UK), washed × 3 in PBS, blocked in PBS containing 0.1% Triton X-100 and incubated with monoclonal anti-βIII-tubulin antibodies (Sigma; diluted at 1:200), to detect DRGN soma and neurites, as described previously^{38 (link),76 (link)}. DRGN were then washed in PBS and incubated secondary antibodies labelled with Alexa-Fluor 488 (Invitrogen). After final washes in PBS, chambers were removed and coverslips mounted with Vectamount containing DAPI (Vector Laboratories, Peterborough, UK). Cells were viewed, by an observer masked to the treatment conditions, under a Zeiss epi-fluorescent microscope equipped with an Axiocam HRc and running Axiovision Software (all from Zeiss, Hertfordshire, UK). Negative controls including omission of primary antibody were included in all experiments and were used to set the background threshold prior to image capture.