Microscopies

download as a pdf protocol DAY 01 Fixation 4% PFA for 30 minutes Wash with PBS Incubate with 15% sucrose and 0.03% Eosin from the stock overnight until they sink at 4C DAY 02 prerequisites pre-freeze isopentane at -80C (it is normally stored at 4C due to its low boiling point) Good to know before starting isopentane is being re-used - when finished embedding, collect used isopentane to designated bottle, try to prevent crystals from getting to the collection bottle isopentane is toxic, hence contact with sewage water is forbidden - only chemical waste can be used for disposal there are three sizes of molds (small, medium, big) - for EHTs we used the middle ones, but the small ones might also be usable based on the sample size. The big ones are too big for EHTs, these are used e.g. for spinal cord samples. DAY 02 Prepare dry ice and isopentane -> How? ...

Download as a pdf Day1 Stock Solutions PBS 4% PFA 1% Tween-20 in PBS 1% Triton X-100 in PBS 5% BSA in PBS antibodies Solutions to be used PBS, used 6x Permeabilization Buffer (Day1, 0.1% Triton X-100, 1% BSA in PBS), used 1x Blocking/primary antibody incubation Buffer (Day1, 0.05% Tween-20, 1% BSA in PBS), used 2x Washing Buffer/secondary antibody incubation buffer (Day2, 0.05% Tween-20 in PBS), used 3x Primary antibodies, used 1x Secondary antibodies, used 1x Volumes 1 well is 300$\mu$L volume (for washing and antibody incubation) Cells fixation move to chemical cabinet in genomic room aspirate media wash with PBS fix with 4% PFA for 10 min. at RT aspirate wash with PBS aspirate add PBS for storage store at 4$^\circ$C Incubation with primary antibodies Permeabilze with 0.1% Triton X-100 in PBS for 15 minutes at RT. Remove permeabilization solution. Wash 1 x with PBS. Add blocking solution (1% BSA in 0.05% Tween-20 PBS) and incubate for 30 minutes. Aspirate the blocking solution. Add diluted antibodies in (1% BSA in 0.05% Tween-20 PBS) overnight at 4$^\circ$C Day 2 Solutions to prepare 0.05% Tween 20 in PBS (for 3 washes and antibody incubation) Solution of secondary antibody in 0.05% Tween 20 in PBS (if there are more secondary antibodies mix them together) DAPI in PBS (1:5000) (make 10mL stock) PBS Mowiol Process Aspirate primary antibody. Wash with 0.05% Tween 20 in PBS (3x) Prepare secondary antibody(ies) solution Add secondary antibody to the wells Incubate for 1h with secondary antibody Aspirate Wash 1x with PBS Add DAPI working solution to stain nuclei Incubate 5min Aspirate Wash 3x 5 min with PBS Check under microscope Mount in Mowiol Store at 4$^\circ$C in dark protocol END ...

Download as a pdf Manufacturer Supplied Protocol Fix and permeabilize the samples as follows: Tissue slices: Use 0.5% Triton X-100 in PBS for 10 minutes. Cells: Use 0.2% Triton X-100 in PBS for 5 minutes. Wash the samples three times with PBS. Blocking and antibody incubation Incubate the samples with a blocking solution for 1 hour at 37°C in a humidified chamber. Prepare the primary antibodies in antibody diluent and incubate the samples overnight at 4°C. Secondary Antibody Staining Wash the samples twice with Buffer A for 5 minutes each. Incubate the samples with PLA probes (anti-mouse MINUS and anti-rabbit PLUS) prepared in antibody diluent for 1 hour at 37°C. PLA reaction Wash the samples twice with Buffer A for 5 minutes each. Perform the ligation step by incubating the samples with ligase enzyme diluted in ligation buffer for 30 minutes at 37°C in a humidified chamber. Wash the samples twice with Buffer A for 5 minutes each. Incubate the samples with polymerase enzyme diluted in amplification buffer for 100 minutes at 37°C in a humidified chamber. Wash the samples twice with Buffer B for 10 minutes each. Perform a final wash with 0.01% Buffer B for 1 minute. Mounting Mount the slides with Duolink in situ mounting medium containing DAPI. Imaging and Storage Wait 15 minutes before imaging using a fluorescence or confocal microscope with at least a 20x objective. After imaging, store the slides: In the dark at 4°C for up to 4 days. Or at -20°C for up to 6 months. Control Experiments Perform negative control experiments by incubating the samples with only one or none of the primary antibodies before adding the PLA probes. ...

Dowload as a pdf Materials EdU kit baseclick: -20◦C; 2.14; ;chemicals box EdU kit baseclick: 4◦C; 2.14; 4F44; kitsBoxes PBS 4% PFA Day1 EdU preparation thaw DMSO diluted stock (10mM) dilute in PBS to 1mM -> working solution Cells labelling add working solution in ratio 1:100 in the wells with media incubate 30 min. at 37◦C Cells fixation move to chemical cabinet in genomic room aspirate media wash with PBS fix with 4% PFA for 10 min. at RT aspirate wash with PBS aspirate add PBS for storage store at 4◦C Cell labelling EdU kit Aspirate PBS Permeabilze with 0.1% Triton X-100 in PBS for 20 minutes at RT Prepare the EdU staining solution (should be used within 15 min) Remove permeabilization solution Wash 2 x with 3% BSA in PBS Aspirate Add EdU staining solution incubate for 30 minutes in dark From now on avoid exposure to light Remove EdU staining solution Wash 3 x with 3% BSA in PBS Aspirate Add PBS Note: You can stop here and store the cells at 4◦C Staining with primary antibodies Aspirate PBS Add blocking solution (1% BSA in 0.05% Tween-20 PBS) and incubate for 30 minutes Aspirate Add antibodies diluted in 1% BSA in 0.05% Tween-20 PBS Keep in dark at 4◦C overnight Day 2 Note: Limit exposure to direct light ...

Flou-4AM MW =1000 dissolve in DMSO to 1-5mM; use 2.5uM link Protocol 24 hours before, change media 30min before acquistion start heating confocal 30 min before acquisition, load with 2.5uM or (5uM) Fluo-4 (search for molarity), I use normal RPMI + B27 but some without phenol red would be even better 15min before acquisition move to 37C confocal, Heating on, CO2 on Find the beating clusters/cells positions using BF and save positions For imaging set linescan 500 per sec 256x256 px binned2x, pinhole 100, laser power 2-8, in BF find the spot put the arrow in the middle do the best focus with BF switch to LIVE, linescanning linescan if it looks okey then do 15 sec at 500fps

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