Immunohistochemistry (PFA perfusion fixed frozen sections) - S100 antibody [4C4.9] - Astrocyte Marker (ab4066)

ab4066 at a dilution of 1/200, staining S100 in astrocytes (Alexa 488 secondary at 1/2000) on 30µm coronal rat brain tissue sections in free floating IHC (see protocol link for detailed description). Image shows astrocytes observed with 20x objective. Image coloured in Photoshop.

NB: No labeling observed following omission of primary antibody.

Sections were viewed using an Axioplan 2 Imaging microscope (Imaging Associates) fitted with 10x, 20x and 40x Plan-Neofluorobjectives (Zeiss, Germany) and images were taken using a AxioCam Hrm digital camera (Zeiss, Germany) and AxioVision software (Imaging Associates).

Immunohistochemistry (Frozen sections) - S100 antibody [4C4.9] - Astrocyte Marker (ab4066)

ab4066 at a 1/200 dilution staining rat spinal cord tissue sections by Immunohistochemistry (Frozen sections). Rats were transcardially perfused with 4% PFA, the tissue was post fixed 1 hour in 4% PFA then 30% sucrose for three days. 20um sections were cut with cryostat. The antibody was incubated with the tissue sections for 18 hours and then bound antibody was detected using an Alexa Fluor ® 555 conjugated goat polyclonal antibody.

This image is courtesy of an Abreview submitted by Nancy Nutile-McMenemy

Immunocytochemistry/ Immunofluorescence - S100 antibody [4C4.9] - Astrocyte Marker (ab4066)

ab4066 at a 1/100 dilution staining rat cortical preps grown 14 days in culture, plated onto coverslips. The preps were acid/alcohol fixed for 15 minutes, blocked and then incubated with the antibody for 1 hour. Bound antibody was detected using a Alexa fluor ® 555 conjugated goat polyclonal antibody. Nuclei were visualised using DAPI (blue). Note: s100 is colocalizing around the nucleus.

This image is courtesy of an Abreview submitted by Ms Nancy Nutile-McMenemy

Western blot - S100 antibody [4C4.9] - Astrocyte Marker (ab4066)

All lanes : Anti-S100 antibody [4C4.9] - Astrocyte Marker (ab4066) at 1/200 dilution

Lane 1 : 40ug Rat spinal cord homogenate
Lane 2 : 40ug Rat spinal cord homogenate
Lane 3 : 40ug Rat spinal cord homogenate
Lane 4 : 40 ug Mouse spinal cord homogenate
Lane 5 : 40 ug Mouse spinal cord homogenate
Lane 6 : 40 ug Mouse spinal cord homogenate
Lane 7 : 40 ug Rat brain homogenate
Lane 8 : 40 ug Rat brain homogenate

Secondary
HRP conjugated goat anti-mouse
developed using the ECL technique

Performed under reducing conditions.

Observed band size : 15,18,21 kDa (why is the actual band size different from the predicted?)


Exposure time : 2 minutes

The extra band at 18kDa may be a result of the protein's thermolabile properties. The band at 21 kDa maybe a dimer of s100a and s100b and the 10/15 kDa may be the subunits. However, there are also references to suggest these may be immunoreactive proteins with differing molecular weights depending on proliferative or differentiated state of the cell (Anat Embryol (Berl). 1997 Nov;196(5):403-16).

This image is courtesy of an Abreview submitted by Ms Nancy Nutile-McMenemy

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - S100 antibody [4C4.9] - Astrocyte Marker (ab4066)

ab4066 staining human melanoma by IHC-P.