EL NUEVO CONTEXTO INTERNACIONAL 177Estados Unidos en Medio Oriente

Individual hypothalami were dissected from the brain tissue and homogenised and sonicated by Katie Burton in 400µl radio-immunoprecipitation assay lysis buffer with protease inhibitor (Sigma-Aldrich), PMSF (Sigma-Aldrich) and PhoSTOP (Roche). Samples were placed on ice and centrifuged at 12000g for 10 minutes. Lysate supernatants were separated into 100µl aliquots and frozen at -80°C until further use. Using the Pierce BCA Protein Assay kit (Thermo Scientific, 23227), protein concentration was quantified according to the manufacturer’s instructions.

97

6.3.3 Immunofluorescence

Brain tissues were carefully cut to isolate the midbrain section of interest. Sectioning of adult Gnasxlm+/p- _{and wildtype mice brains was performed on the Leica CM 1950 Cryostat at -20°C}

by Katie Burton, using Shandon Cryomatrix mounting media (Thermo Scientific). Frozen coronal sections at a thickness of 14µm were transferred to SuperFrost Plus slides (Thermo Scientific) and stored at -80°C until further use. Sectioning of postnatal day 1 and postnatal day 5 mice (P1 and P5 respectively) for both Gnasxlm+/p- _{and wildtype mice brains was}

performed by myself, using the same method.

Slides were incubated in 10mM sodium citrate for one minute at 60°C; washed three times in PBS at room temperature for 5 minutes; blocked for one hour at room temperature in 10% donkey normal serum in PBS; and incubated overnight at 4°C in 10% donkey normal serum in PBS, 0.25% triton, 1:500 Polyclonal rabbit anti-GFAP primary antibody (Dako Z0334) in PBS. Slides were then washed three times for 15 minutes in PBS at room temperature. These were incubated in 10% donkey normal serum in PBS, 0.25% triton, 1:1000 4’,6’-diamidino-2- phenylindole (DAPI) and 1:1000 donkey anti-rabbit IgG Alexa488-tagged secondary antibody for one hour at room temperature in the dark. Slides were washed three times in PBS for 15 minutes prior to mounting with Fluoro-gel (Electron Microscopy Sciences), drying, and sealing with clear nail varnish. Slides were stored in the dark at 4°C.

6.3.4 Microscopy

Epifluorescence microscopy was performed using the Zeiss Axioskop 40 microscope. Images were taken and processed using the AxioVision 4.6 software (Zeiss).

6.3.5 Self-cast SDS-PAGE

6.3.5.1 Anti-GFAP antibody incubation

After transferring, the membrane was carefully removed and marked in the top corner to mark the side on which the protein adhered to. The membrane was washed in PBS prior to blocking in PBS Tween with 5% non-fat dried milk powder for one hour at room temperature. Membranes were sealed in a plastic bag and incubated with 2ml 1:6000 polyclonal rabbit anti-GFAP primary antibody (Dako Z0334) in blocking solution (PBS Tween and 5% non-fat dried milk powder) for one hour at room temperature on a shaker. Membranes were washed four times for five minutes each in PBS Tween prior to a one hour incubation in a sealed plastic bag with 10ml 1:10000 donkey anti-rabbit horseradish peroxidise (HRP)-tagged secondary antibody. Membranes were washed again in PBS Tween four times for five minutes each to remove excess antibody.

98

6.3.5.2 Enhanced chemiluminescence detection and exposure to film

Enhanced chemiluminescence (ECL) was performed, in order to detect presence of the HRP- tagged secondary antibody. This was achieved using the Amersham ECL Plus Western blotting reagent pack (GE Healthcare), according to the manufacturer’s instructions. Briefly, excess wash buffer was drained prior to incubating the membranes in a 40:1 ratio of solution A to solution B on the protein side of the membrane, for five minutes. Membranes were then drained of excess ECL reagent and wrapped protein side down in Saran wrap. A fluorescent marker was attached to the front of the Saran wrap so that after exposure onto film, it would be possible to transfer the bands of the protein ladder to the film in order to estimate the densities of proteins within the bands present on the membranes. These were then placed protein side up in a film cassette in the dark room. The length of exposure time was varied, depending on the strength of the signal from the blot. After exposure, the film was placed into a tray of developing solution (Kodak) for one minute, followed by a tray of fixer solution (Kodak) for one minute, before being placed into a tray of water and then hung to dry. After drying, the protein ladder was marked on the film by overlaying the film on top of the blot using the position of the fluorescent marker.

6.3.5.3 Anti-β-actin antibody incubation

β-actin and GFAP have similar molecular masses (42kDa and 50kDa, respectively). In addition, both of these primary antibodies were raised in rabbits. Therefore it was necessary to strip the membrane of both primary and secondary antibodies.

Stripping was performed using a buffer containing 100mM β-mercaptoethanol, 62.5mM Tris- HCl (pH 6.7), and 2% SDS. This was mixed under the fume hood, and decanted into an airtight container with the membranes and placed in a shaking water bath pre-heated at 60°C, for 30 minutes. After stripping, the solution was poured away under the fume hood, and the membranes were washed in PBS Tween using large volumes of wash buffer, twice for 10 minutes each. This is to ensure removal of the β-mercaptoethanol.

Incubation with the polyclonal rabbit anti-actin primary antibody (Santa Cruz C11 sc-1615-R) was performed using the same incubation times and washing steps as for GFAP previously. However, the β-actin primary antibody was used at 1:1000 in blocking solution. After incubation with the same HRP-tagged secondary antibody as was used for GFAP, the membranes were subjected to ECL reagent and film exposure, as previously.

99

6.3.6 NuPAGE SDS-PAGE

The XCell SureLock Mini-Cell Electrophoresis System (Life Technologies) was used to perform Western blots, in conjunction with NuPAGE Novex 4-12% Bis-Tris 1.5mm, 10 well gels (Life Technologies). Samples were heated to 70°C for 10 minutes, after which reducing agent was added (as in manufacturer’s protocol). Gels were run using MOPS buffer with antioxidant (Life Technologies). Samples were run with the Spectra Multicolor Broad Range protein ladder (Thermo Scientific), which shows apparent molecular weights of 225, 115, 80, 65, 50, 35, 30, 25, 15 and 10 kDa when run on this gel system in MOPS buffer. Gels were run for 50 minutes at 200V, according to the manufacturer’s suggestion.

6.3.6.1 Blotting to membrane

Blotting was performed using the XCell II Blot Module (Life Technologies) and self-made NuPAGE transfer buffer, consisting of 25mM bicine, 25mM bis-tris, 1mM EDTA (Sigma- Aldrich), 0.05mM chlorobutanol (Sigma-Aldrich), with 10% methanol (Fisher Scientific). 20% methanol was used when running two gels in the same tank, according to the manufacturer’s suggestion, to enable sufficient transfer of protein to both membranes. Amersham Hybond- P PVDF membrane (GE Healthcare) was pre-wetted in methanol for 10 seconds, washed in deionised water for five minutes. Phosphate buffered saline (PBS) was made by dissolving one PBS tablet (Sigma-Aldrich) in 200ml of deionised water. The PBS composition was 0.01M phosphate buffer, 0.0027M potassium chloride and 0.137M sodium chloride at pH 7.4. Membranes were equilibrated in PBS with 0.1% Tween 20 (Sigma-Aldrich) for 10 minutes prior to transfer. The blot module was set up such that the wetted membrane was placed on the anode side of the gel, surrounded by 3MM filter paper (Whatman) and blotting pads, all saturated in transfer buffer. The blot module was filled with NuPAGE transfer buffer containing antioxidant. Blots were run at 30V for one hour, as suggested by the manufacturer. After transfer, membranes were placed between 3MM filter paper (Whatman), wrapped in Saran wrap, and stored at 4°C overnight.

6.3.6.2 Antibody incubations

GFAP antibody incubation was performed as previously when using self-cast SDS-PAGE gels. However, the volume of polyclonal rabbit anti-GFAP primary antibody (Dako Z0334) in blocking solution was increased from 2ml to 5ml to ensure complete saturation of the membrane.

100

Membrane stripping was also performed as previously, with the incubation time reduced to five minutes. β-actin antibody incubation, ECL detection, and film exposure were performed identically as previously described.

6.3.6.3 Quantification of GFAP

Quantification was performed using image processing software, ImageJ (NIH). The tool allows for quantification of gels. This is achieved by analysing rectangular selections of the image to allow analysis lane by lane. The intensity of the bands are then converted to a graph, displaying peaks of intensity. By using the straight line tool, a line is drawn across the base of the peaks, and the wand tool is used to measure the area between the peaks and the straight line. The area measured on the GFAP gel image was then divided by the area measured from the β-actin image, for each sample. This allowed GFAP levels for each sample to be normalised to the β-actin control.

6.4 Results

RNAseq and qPCR are well established techniques for quantification of mRNA. However, for protein-coding genes, it is the protein which plays the active functional role, not the RNA. Since RNA expression does not always correlate with protein levels, understanding protein expression and localisation is vital in ameliorating the physiological changes that occur in the hypothalamus in Gnasxlm+/p- _{mice. Therefore, after identifying a decrease in Gfap by RNAseq}

and qPCR analyses, it was important to investigate whether the same was true of GFAP protein and how its expression and localisation is affected in Gnasxlm+/p- _mice.