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Roman - Optical M 42622.pptx
- 1. Mouse Spinal Cord Immunofluorescence Andrew Roman BIOL 6222 Dr. Michael Rea, Dr. Kathleen Gajewski 4/26/2022
- 2. Introduction Figure 1. Lumbar region of the mouse spinal cord (Sengul et al., 2012). • Spinal cord is divided into lamina (Caspary et al., 2003) • Network of neurons connect in a translaminar fashion (Johannssen et al., 2013) • Cell types are classified using molecular markers (Dobrott et al., 2019). • Indirect immunofluorescence can determine the localization of a protein in a cell (Donaldson, 2015) Figure 2. Sensory neurons project onto specific laminae while motor neurons connect directly to muscles (Caspary et al., 2003).
- 3. Hypothesis Figure 3. The roles of LXR signaling are cell type specific (Courtney et al., 2016) • If LXR is present in the same cell as marker of known location, then that cell expresses LXR. • If a cell is double-labeled, then it will produce a yellow signal. • If a cell does not contain LXR, then it will be red. • If LXR is expressed in a GFAP labeled astrocyte, then there will be a yellow signal within the cell or on its surface. • If LXR is expressed in an Iba1 labeled microglial cell, then a yellow signal will be observed on or inside the cell. • If LXR is expressed in a NeuN labeled neuron, then a yellow signal will be observed. • If LXR is expressed in a Olig2 labeled oligodendrocyte, then a yellow signal will be observed. Figure 4. Comparison of direct and indirect immunofluorescence (Abcam, 2021)
- 4. Materials and methods 1. Labeled a slide with the name of the protein marker and date 2. Collected data from paraffin sections of female spinal cord • 11M wild-type • 29M wild-type female 3. Deparaffinized the tissue, retrieved the antigen with PT module heated bath 4. Made a well around the tissue with PAP pen 5. Utilized 0.5% Triton solution for LXR nuclear staining 6. Primary antibody solutions in donkey serum • anti-Olig2 1:600 • anti-GFAP 1:1000 • anti-Iba 1:1000 • anti-NeuN 1:1000 • anti-LXRs 1:2000 • anti-LXRβ 1:2000 7. Secondary antibody solution 1:400 in PBST • Anti-Rb Green 488 (LXRs) • Anti-Gt Green 488 (LXRβ) • Anti-Ms Red 594 (GFAP, NeuN) • Anti-Rb Red 594 (Olig2, Iba1) 8. DAPI and coverslip, sealed with nail polish 9. Examined slides on Olympus FV3000 inverted confocal microscope • 20X UCPLFLN20X N/A 0.7, WD 0.8-1.8 mm, w/Correction Collar Product Name Species Brand & Product Number anti-Olig2 rabbit Abcam ab-109186 anti-Iba1 rabbit Abcam ab-178846 anti-GFAP mouse Santa Cruz sc-65343 anti-NeuN mouse Millipore MAB377 anti-LXRbeta goat homemade anti-LXRs rabbit LifeSpan Biosci. LS-B262 anti-goat 488 donkey ThermoFisher Alexa Fluor A11055 anti-mouse 594 donkey ThermoFisher Alexa Fluor A21203 anti-rabbit 594 donkey ThermoFisher Alexa Fluor A21207 DAPI mounting medium Santa Cruz sc-24941
- 5. Results • NeuN stained cells showed some puncta with LXR • GFAP stained cells had no LXR immunoreactivity • Few or no Olig2 stained cells showed nuclear LXR staining • Some Iba1+ cells showed nuclear LXR immunoreactivity
- 10. Olig2, LXR, DAPI
- 11. Olig2, LXR, DAPI
- 12. Iba1, LXR, DAPI
- 13. Iba1, LXR, DAPI
- 14. Iba1, DAPI
- 15. Abcam. (2021, January 21). Direct vs indirect immunofluorescence. https://www.abcam.com/secondary-antibodies/direct-vs- indirect-immunofluorescence Caspary, T., & Anderson, K. V. (2003). Patterning cell types in the dorsal spinal cord: what the mouse mutants say. Nat. Rev. Neurosci., 4(4), 289-297. https://doi.org/10.1038/nrn1073 Courtney, R., & Landreth, G. E. (2016). LXR regulation of brain cholesterol: from development to disease. Trends Endocrinol. Metab., 27(6), 404-414. https://dx.doi.org/10.1016%2Fj.tem.2016.03.018 Dobrott, C., Sathyamurthy, A., & Levin, A. J. (2019). Decoding cell type diversity within the spinal cord. Current Opinion in Physiology, 8, 1-6. https://doi.org/10.1016/j.cophys.2018.11.006 Donaldson, J. G. (2015). Immunofluorescence staining. Current protocols in cell biology, 69(1), 4- 3. https://doi.org/10.1002/0471143030.cb0403s69 Johannssen, H. C., & Helmchen, F. (2013). Two-photon imaging of spinal cord cellular networks. Experimental Neurology, 242(), 18-26. https://doi.org/10.1016/j.expneurol.2012.07.014 Sengul. G., & Watson, C. (2012). Spinal cord. In C. Watson, G. Paxinos, & L. Puelles (Eds.), The mouse nervous system (pp. 424- 458). Academic Press. Works Cited
Editor's Notes
- Good morning everyone, my name is Andrew and I am presenting my project on localizing proteins in the mouse spinal cord using immunofluorescence and the confocal microscope.
- The mouse central nervous system consists of the brain and spinal cord. The other nerves in the body are the peripheral nervous system. The spinal cord is divided into 34 segments, consisting of cervical, thoracic, lumbar and coccygeal sections. Each segment sections each containing nerves that innervate different muscles. For example, the nerves in the L1 region of the spinal cord FIG1 appear to be involved with the flexing and extending of muscles. The peripheral nervous system sends signals to the spinal cord from specialized cells. These cells send projections through the dorsal root ganglia into the different lamina of the spinal cord. In FIG2 we see how these nerves feed into the dorsal horn of the spinal cord. Networks of neurons spread throughout the lamina in a translaminar fashion through axonal projections. The motor neurons on the other hand project out of the ventral spinal cord onto muscles. Different cell types can be identified using molecular markers. A method called indirect immunofluorescence can be used to determine if a protein is located within a cell.
- This method of indirect immunofluorescence uses multiple antibodies to label an antigen. When the location of one protein is known a second marker may be added to the tissue in order to localize the unknown protein. By labeling the cells with LXR and markers for astrocytes, microglia, neurons, and oligodendrocytes the aim of this experiment was to determine which cells in the mouse spinal cord are highly IR for LXR. Ablation of LXRs, notably LXR beta results in a neurodegenerative phenotype in knockout mice. This ALS-like loss of motor neurons is combined with accumulation of lipids according a 2005 article by Gustafsson etal. Although dysregulation of lipid homeostasis is assoicated with serveral ND diseases, there is no known mechanism for the death of MN. LXR controls the efflux of cholesterol in cells and regulates AQP4, and TFs like ABCA1. Astrocytes produce cholesterol which is used by neurons to maintain synapses. LXR is activated by the cholesterol metabolite 24-OHC. Microglia and astrocytes produce and lipidate apoE. For this experiment, it was hypothesized that if nuclear LXR were present within a cell containing a known marker, then it would generate a yellow signal on the fluorescence microscope. Astrocytes were labeled with glial fibrillary acidic protein, microglia with Iba1, neurons with NeuN, and oligodendrocytes with Olig2.
- The mouse spinal cord sections that were used came from an eleven month-old wild type female and a 29 M WT female. The sections were made from paraffin blocks containing a small piece of tissue from the L1 portion of the spinal cord. Xylene and alcohol were used to deparaffinize and rehydrate the tissue. A warm citrate buffer bath inside of a PT module was used to unmask the antigens. Using a wax pen a well was made around the section. Some triton was added to the section in order to permeablizie the nuclear membrane. If this is not done then the LXR antibody will not reach the cell nucleus. After the triton was washed off a solution of primary antibody was added to the tissue and allowed to incubate for an hour. This solution was washed off and the secondary antibody was added. The secondary antibody contains the fluorophore that illuminated the target antigen. LXR was labeled in green and the known markers Iba1 etc were labeled in Red. The last step is to add a drop of DAPI and a coverslip. Then the slide was sealed with nail polish and examined on the Olympus FV3000 inverted confocal microscope using a 20X objective which is equipped with correction collar. Our lab does not normally use #1.5 coverslips so the correction collar was adjusted in order to visualize the tissue sections at this magnification. The notch on the correction collar is shown in the picture. The table shows the different products I used to stain the tissue, what species they were made in, and the manufacturer.
- After examination of this tissue, it was concluded that the NeuN+ neurons showed some puncta in the cytoplasm, GFAP+ cells had no LXR IR, Olig2+ cells had little or no LXR, and Iba1+ cells were highly IR. In the future, I would like to utilize DAPI that does not contain mounting media and #1.5 coverslips in order to examine the staining at higher magnification. Furthermore, I plan to examine the male mice, compare the expression of LXR to the female, and stain some brain tissue as a positive control.
- This micrograph shows a NeuN stained neuron.
- Although there is some green anti-LXR it does not appear in the nucleus, however, there are some puncta in the cytoplasm.
- This micrograph shows some red GFAP+ astrocytes in the spinal cord
- The LXR antibody does not appear in the nucleus or the axonal processes.
- This is the Olig2 staining
- There may be one yellow cell here but the nuclear DAPI staining does not appear to overlap with this area. This could a dead cell or some non-specific staining.
- This is an Iba1+ microglia. Here you can see how the DAPI, red and green stains overlap indicating that the Iba1 antibody and LXR antibody are within the same cell.
- This is another Iba1+ microglia showing similar results.
- This an Iba1 immunostaining. It shows some of the cellular structures that branch out from the cell's main body.
- I consulted these sources for this presentation.