2.4. Immunolocalization
To investigate the effects of animal feeding behaviour with this
technique, samples from the same 8 animals that were studied during the
western blotting analysis were utilised. For each animal, one 5
cm2 section of ventral sac rumen tissue was fixed
overnight in 4% (w/v) paraformaldehyde dissolved in PBS, and then
embedded in paraffin wax. These paraffin-embedded rumen tissues were
then cut into 5 µm sections and attached to positively charged
microscope slides (SuperFrost Ultra Plus, Thermo Scientific). After
overnight drying of these sections, paraffin was removed using
Neo-clear® treatment and sections rehydrated through a series of
descending alcohol concentrations (100%, 95% and 70%). Endogenous
peroxidase activity was blocked with 0.3%
H2O2 in methanol. Quenching was
performed with 50 mM NH4Cl in phosphate buffered saline
(PBS). The sections were then washed with 1% BSA, 0.2% gelatin, and
0.05% saponin in PBS. Next, sections were incubated in either 1:250
UT-Bc19 or 1:2000 anti-MCT1 antibodies diluted in 0.1% BSA and 0.3%
Triton X-100 in PBS, or in buffer only (i.e. control), overnight at
4oC. After warming at room temperature for 1 hour,
sections were washed with 0.1% BSA, 0.2% gelatin, and 0.05% saponin.
Sections were then incubated for an hour with the secondary horseradish
peroxidase-conjugated anti-rabbit or anti-chicken antibodies diluted to
a concentration of 1:200 with 0.1% BSA, 0.3% Triton X-100 in PBS.
Next, sections were washed again with 0.1% BSA, 0.2% gelatin, and
0.05% saponin in PBS solution. For detection, freshly made
3,3’-diaminobenzidine (DAB) was added onto the sections for 10 minutes.
The sections were then rinsed in PBS to stop the reaction and
counterstained with haemotoxylin. The stained sections were then
dehydrated through a series of increasing alcohol concentration (70%,
95% and 100%), followed by Neo-clear® and coverslips mounted using
Eukitt mounting medium. Finally, detailed images of sections were
obtained using a light microscope (Leica, Germany) and a digital camera
(DFC420C, Leica, Germany) with its software (Application Suite V4).