1. Immunofluorescence and In-situ Hybridisation Shameem Ladak, PhD : s.s.Ladak@newcastle.ac.uks.s.Ladak@newcastle.ac.uk Nina Jordan, PhD : nina.jordan@newcastle.ac.uknina.jordan@newcastle.ac.uk

2. Immunofluorescence NINA JORDAN

3. Using molecule with luminescent properties In molecule, electrons have a stable level of energy Excitation with light Electron absorbs energy from photon (less stable energy level) Electron release energy in a new photon Emitted fluorescence Excited state Ground state Excitation Emission Principle of fluorescence

4. Immunofluorescence direct and indirect There is two type of immunofluorescence Direct immunofluorescence Primary antibody conjugated Faster Reduced cross-reactivity Reduced non-specific binding Indirect immunofluorescence Secondary antibody conjugated Cheaper More flexible Amplified signal

5. Protocol of immunofluorescence

6. Set up your experiment ControlWhy? Preparation Autofluorescence Do my cells release basal fluorescence? No antibody added Secondary antibody (only indirect IF) Does my secondary antibody bind to something else than my primary antibody No primary antibody Multicolor IF Does my primary antibodies affect each other in antigen binding ? Single staining for each fluorochrome Blocking peptide (more optional) Does my first antibody bind to my antigen? Add blocking peptide before primary antibody

7. Fixation and permeabilisation + Preserve cellular morphology - Antigen might be crosslinked Organic solvents Methanol or Acetone Fixation and permeabilisation Chemical Crosslinkers Formaldehyde or Glutaradehyde Only fixation + Preserve cellular architecture - Soluble and lipid component lost Additionnal permeabilisation step: Triton X-100 /NP-40 /Saponin /Tween20 /Digitonin

8. Blocking and antibodies incubation Blocking solution BSA,milk or serum in PBS is used to block the non-specific binding of the primary antibody. Do not use the same specie for the serum than specie of the primary antibody Incubation at room temperature: 30 minutes to 1 hour First antibody Dilution in the blocking solution Incubation overnight at 4Cº Wash three time with PBS (5min) Second antibody Against the specie of the primary antibody Dilution in the blocking solution Incubation from 1 to 2 hour at room temperature in the dark Wash three time with PBS (5min) Washes: Drop PBS and remove it carefully

9. Nucleus staining and mounting DAPI diluted in PBS Incubation 15 to 30 min Wash three times with PBS(5min) Add the mountant medium Or Mountant medium containing DAPI A small drop of mounting medium is placed on the sample. A coverslip is carefully lowered onto the drop of mounting medium in order to prevent the formation of bubbles. Drop the mounting medium on the coverslip and not the sample = less bubble

10. Microscope Set up gain and exposure each fluorochrome on the microscope. Too much exposure can photobleach your sample. Begin with a minimum exposure and increase it if needed Set up the exposure first on your control without primary to ensure that your staining is specific.Then move with the staining Gain will increase the brigthness of your staining. Gain and exposure should not be change between slides in order to compare and quantify your results

11. My protocol for immunofluorescence of cells Fixation in cold methanol during 10min Wash one time with PBS 5min Blocking one hour on the rocker with 1%BSA in PBS Primary antibody dilution 1/100 in 1%BSA in PBS, Incubation overnight Wash two times with PBS (5 min on rocker) Secondary antibody dilution 1/100 in 1%BSA in PBS Incubation one hour on rocker Wash two times with PBS (5 min on rocker) Mount the slides with mounting media containing DAPI

12. Trouble shooting 1)No staining The primary antibody and the secondary antibody may not match. Secondary antibody needs to be against the specie of the primary antibody Protein is non abundant in the sample, run a positive control Fixation method degrade the protein of interest 2) Staining non specific Concentration of antibodies could be too high Slides could be not washed properly Secondary antibody non specific 3) Dried slides Do not let the slide dry out, use humidified chamber or sealed with nailed polish in order to re-use the slides 4)Photobleaching Do not expose your slide for a long time under the light. Keep your slides in the dark. Fluorescence can be lost easily

13. Analysis ImageJ software Quantification in two step -Count number of cells -Quantify area of fluorescence for each fluorochrome Calculate a ratio of the quantification of each fluorochrome on the number of cells to obtain fluorescence per cells Analysis of three pictures minimum per slide to calculate statistics

14. In-situ hybridization SHAMEEM LADAK

15. Principle It is a method of localizing and detecting specific RNA sequences in morphologically preserved tissues sections or cell preparations. The most common tissue sections used with in situ hybridization Frozen sections, Paraffin embedded sections and Cells in suspension (fixed with methanol on glass slides)

16. Background and Introduction Common Methods to Detect and Quantify miRNA 1.Northern blotting, qRT-PCR and Microarray fail to locate the miRNAs within tissue or cells. 2.Evaluation of the spatial expression of miRNAs is especially crucial for those tissues with a number of different cell types. 3.In 2004, in situ hybridization (ISH) technology was used for the first time in plants to assess miRNAs 4.LNA probe: ribose ring is “locked” in the ideal conformation for Watson-Crick binding. Result: rapid binding to complementary strand & increased thermal stability

17. Fluorescence in situ hybridization (FISH) for microRNA detection DIG-labeled in situ hybridization for microRNA detection

18. Workflow overview: One day miRNA ISH protocol Total time required (with washes): 9 hours

19. Deparaffinize slides in Xylene and ethanol Permeabilization of cells and tissues- proteinase K Dehydrate slides- Ethanol Hybridization with LNA probes Incubation with anti-DIG reagent Incubation with AP substrate Counter stain- Nuclear stain Dehydrate and mount slides Microscopy 20 minutes Steps 26 minutes (includes PBS wash and dehydration ) 1 hour and 45 minutes ( includes washes and blocking step) 1 hour and 10 minutes (includes washes) 1 hour and 45 minutes (includes washes) Time 6 minutes ( includes washes) 5 minutes ( includes washes) Total time: 5 hours and 37 minutes

20. Optimization 1.Proteinase K treatment and hybridization temperature: using housekeeping gene (LNA u6 snRNA) Tip 1: Adjust concentration (5-20 μg/mL, 10mins, 37°C) and duration (5-30 minutes, 15μg/mL, 37°C) of Proteinase K Tip 2: Identify ISH sensitivity level (using dilutions of the LNA™ U6 snRNA probe) Tip 3: Identify optimal hybridization temperature

21. 2.Control study using the optimized protocol parameters with the LNA™ postive control and LNA™ Scramble-miR negative control probe

22. 3.Finally, detect the microRNA(s) of interest using the appropriate miRCURY LNA™ microRNA Detection probe(s) with the defined protocol parameters Diseased Control miR- LNA -ve control +ve control Manual scoring of ISH Staining intensity grouped as following: 0= negative 1= weak 2=intermediate 3= strong

23. Troubleshooting 1.No signal: Check if reagents were reconstituted/ prepared correctly. 2.Low sensitivity with the LNA™ U6 snRNA probe: Check if reagents were prepared correctly or/and tissue section is optimum. 3.Strong U6 snRNA signal but no or low microRNA signal: sub- optimal Proteinase-K treatment! Optimize it correctly. 4.High background staining: Increase the hybridization temperature and/or no of washes

24. 5.Non-specific staining: Tricky one! Check if non specific binding is due to DIG-labeled probe itself, the detecting antibody or due to endogenous enzymatic reactions. I.If staining is obtained in the absence of AP-conjugated anti-DIG then endogenous AP is present. II.If staining is obtained in the absence of the DIG-labeled probe (and no endogenous AP activity is observed) then staining is related to the detecting antibody. III.If abundant endogenous enzymatic reactivity cannot be prevented by Levamisol, Fluorescence – ISH approach needs to be used. Also check for SSC wash buffer temperatures!

25. 6.Non-specific staining of ECM: Concentration of Anti-DIG antibody is too high. 7.Sections fall off after de-paraffination: Small and thick sections fall off more easily than large thin sections Avoid storage of paraffin sections at -20°C