Sep 11, 2024

Public workspaceParamecium bursaria immunofluorescence protocol

DOI
dx.doi.org/10.17504/protocols.io.yxmvm2ozng3p/v1
  • 1University of Oxford
  • Richards Lab
David S Milner
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DOI: dx.doi.org/10.17504/protocols.io.yxmvm2ozng3p/v1
Protocol Citation: David S Milner, Estelle Kilias, Thomas A. Richards 2024. Paramecium bursaria immunofluorescence protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm2ozng3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 01, 2022
Last Modified: September 11, 2024
Protocol Integer ID: 69436
Funders Acknowledgement:
European Research Council
Grant ID: CELL-in-CELL: 819507
Abstract
This protocol has been developed for immunofluorescent localisation of host proteins in Paramecium bursaria 186b.
Protocol materials
ReagentGoat Anti-Rabbit IgG H&L (Alexa Fluor® 488)AbcamCatalog #ab150077
Step 10
ReagentGoat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbedAbcamCatalog #ab150117
Step 10
Reagent Anti-α-Tubulin antibody Mouse monoclonalMerck MilliporeSigma (Sigma-Aldrich)Catalog #T6199-25UL
Step 8
ReagentPhosphate buffered salineMerck MilliporeSigma (Sigma-Aldrich)Catalog #P4417-50TAB
Before starting
ReagentPierce™ 16% Formaldehyde (w/v) Methanol-freeThermo Fisher ScientificCatalog #28906
Step 3
ReagentBovine Serum AlbuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #A2153
Step 7
ReagentCITIFLUOR Antifadent Mounting Medium AF2Electron Microscopy SciencesCatalog #17971-100
Step 13
ReagentTween 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P1379-500ml
Step 5
ReagentHoechst 33342Invitrogen - Thermo FisherCatalog #H1399
Step 11
ReagentPoly-Prep SlidesMerck MilliporeSigma (Sigma-Aldrich)Catalog #P0425-72EA
Step 14
Before start
Prepare 200 mL of PBS

ReagentPhosphate buffered salineSigma AldrichCatalog #P4417-50TAB

Fixation
Fixation
52m
52m
Take 6 mL of P. bursaria culture per treatment. Centrifuge Centrifigation800 x g, 00:10:00 and remove 5 mL (i.e. concentrate sample 6-fold). Aliquot 1 mL of concentrated culture into a 1.5 mL micro-centrifuge tube per treatment for downstream steps.
10m
Centrifuge Centrifigation800 x g, 00:10:00 to pellet the cells. Remove supernatant, avoiding the cell pellet.
10m
Re-suspend cells in Amount500 µL PBS containing 4% formaldehyde [Amount375 µL PBS plus Amount125 µL 16% formaldehyde]. Use a fresh ampule of formaldehyde for each experiment.

Incubate for Duration00:12:00 TemperatureRoom temperature

ReagentPierce™ 16% Formaldehyde (w/v) Methanol-freeSigma AldrichCatalog #28906

12m
Centrifuge Centrifigation800 x g, 00:10:00 , remove supernatant and wash once in Amount500 µL PBS. Repeat the centrifugation Centrifigation800 x g, 00:10:00

20m
Permeabilisation
Permeabilisation
40m
40m
Re-suspend cell pellet in Amount500 µL PBS containing Concentration0.05 % volume Tween 20.

Incubate for Duration00:10:00 TemperatureRoom temperature

ReagentTween 20Sigma AldrichCatalog #P1379-500ml

10m
Centrifuge Centrifigation800 x g, 00:10:00 , remove supernatant and wash twice in Amount500 µL PBS. Repeat the centrifugation Centrifigation800 x g, 00:10:00 , x2
30m
Blocking
Blocking
1h
1h
Prepare PBS containing Concentration3 % volume BSA. Filter through a 0.2 micron syringe filter prior to use. Re-suspend cells in Amount500 µL of this solution.

Duration01:00:00 TemperatureRoom temperature

ReagentBovine Serum AlbuminSigma AldrichCatalog #A2153

1h
Primary antibody
Primary antibody
16h
16h
Add Amount1 µL primary antibody directly to the cells in PBS+3% BSA.

A good positive control here is Amount1 µL Anti-α-Tubulin antibodyReagent Anti-α-Tubulin antibody Mouse monoclonalSigma AldrichCatalog #T6199-25UL

DurationOvernight Temperature4 °C


Note
You will need to titrate an appropriate concentration of the primary antibody here

16h
Secondary antibody
Secondary antibody
3h
3h
After the overnight incubation, wash three times in Amount500 µL PBS.

Repeat the centrifugation Centrifigation800 x g, 00:10:00 , x3 and re-suspend the cells in Amount500 µL PBS.

30m
Add Amount1 µL secondary antibody.

For rabbit primary antibodies:
ReagentGoat Anti-Rabbit IgG H&L (Alexa Fluor® 488)Sigma AldrichCatalog #ab150077

For mouse primary antibodies (e.g. Anti-α-Tubulin antibody used above):
ReagentGoat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbedSigma AldrichCatalog #ab150117

Duration02:00:00 TemperatureRoom temperature in the dark

2h
If required, add Amount2 µL of Concentration10 mg/mL Hoechst 33342

ReagentHoechst 33342Sigma AldrichCatalog #H1399

Duration00:05:00 TemperatureRoom temperature

5m
Centrifuge Centrifigation800 x g, 00:10:00 , remove supernatant and wash twice in Amount500 µL PBS. Repeat the centrifugation Centrifigation800 x g, 00:10:00 , x2 and re-suspend in Amount20 µL of residual PBS

20m
Preparing slides
Preparing slides
25m
25m
Add a drop of CITIFLUOR AF2 to the residual PBS.

ReagentCITIFLUOR Antifadent Mounting Medium AF2Electron Microscopy SciencesCatalog #17971-100



5m
Using a wide-bore tip (i.e. a 1 mL pipette tip with the end cut-off), spot approximately Amount10 µL ontoReagentPoly-Prep SlidesSigma AldrichCatalog #P0425-72EA
Note
Add only one sample to the centre of each slide to allow for sealing


5m
Add a coverslip and seal with clear nail polish.
15m