Sep 03, 2024
Version 1
Immunofluorescence for detection of ALFA-tag in S. rosetta V.1
- 1UCSF;
- 2University of California, San Francisco
Protocol Citation: Fredrick leon, David Booth 2024. Immunofluorescence for detection of ALFA-tag in S. rosetta . protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl85k38l2w/v1Version created by David Booth
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: May 19, 2021
Last Modified: September 03, 2024
Protocol Integer ID: 104831
Keywords: choanoflagellate, immunofluorescence, ALFA-tag
Abstract
Immunofluorescence (IF) allows for the visualization of protein localization in fixed-cell samples. This protocol builds upon commonly used IF methods, with a focus on retaining and minimally damaging S. rosetta's collar and flagella. It also introduces a new permeabilization buffer composed of LICOR Intercept blocking buffer for greater anti-ALFA tag nanobody specificity and signal. This protocol has been optimized for IF with the ALFA tag, but works well for common antibodies, such as DM1A and phalloidan. This protocol was developed with essential input from Flora Rutaganira and Alain Garcia de las Bayones
Protocol materials
Synthetic Seawater ASTM D 1141Ricca Chemical CompanyCatalog #8363
Step 6
Poly-D-Lysine HydrobromideFisher ScientificCatalog #ICN10269410
Step 5
clear bottomed 96 well platesMP BiomedicalsCatalog #102694
Step 5
INTERCEPT blocking buffer, PBSLI-CORCatalog #927-70001
Step 11
Grow cells
Grow cells
Seed cells at 10,000 cells/mL 24 hours hours in advance and grow at 22 °C
On the day of imaging, gently concentrate cells by centrifugation at 500 x g, 22°C, 00:05:00
5m
Carefully remove supernatant with a pipette, leaving the last 50-100 µL
Note
Leaving behind some supernatant is because the cell pellet is very delicate after the light spin and may not be visible.
Gently swirl the remaining supernatant and cells to resuspend the pellet. Do NOT vortex.
Adsorb cells to Glass-Bottom Dish
Adsorb cells to Glass-Bottom Dish
45m
45m
Pipette 50 µL of 10 mg/mL Poly-D-Lysine onto the glass bottom of a glass-bottomed 96 well plate and incubate for 00:15:00 at Room temperature .
Poly-D-Lysine HydrobromideFisher ScientificCatalog #ICN10269410
clear bottomed 96 well platesMP BiomedicalsCatalog #102694
15m
Remove the excess liquid and add 50 µL filtered Synthetic Seawater (SSW) . Repeat twice more for a total of 3 washes.
Synthetic Seawater ASTM D 1141Ricca Chemical CompanyCatalog #8363
Gently pipette 50 µL cells onto the surface of the glass. Use a wide-bore pipette tip (or cut a 1000 µl pipette tip) to decrease shear forces on cells. Let the cells settle on the surface for 00:15:00 .
15m
Prepare buffers
Prepare buffers
Assemble The following buffers, and use all at room temperate
PFA fix buffer: Fixes the cells initially
| A | B | |
| 100 μL | 10x CSB | |
| 187.5 μL | 16% paraformaldehyde | |
| 250 μL | 60% (w/v) sucrose | |
| 462.5 μL | water | |
| 1 mL | total |
This buffer is made fresh each time
PFA/Tween fix buffer: Continues the fixation process and the Tween addition very gently permeabilizes cells
| A | B | |
| 100 μL | 10x CSB | |
| 187.5 μL | 16% paraformaldehyde | |
| 250 μL | 60% (w/v) sucrose | |
| 3.5 μL | 20% (v/v) Tween 20 | |
| 459 μL | water | |
| 1 mL | total |
This buffer is made fresh each time
Gycline wash buffer : Quenches the remaining PFA from the previous fixation steps
| A | B | |
| 500 μL | 10x CSB | |
| 1500 μL | 1M glycine, ph 6.1 | |
| 1250 μL | 60% (w/v) sucrose | |
| 1750 μL | water | |
| 5 mL | total |
this can be made in bulk and stored at 4°C
PermB-meOH buffer: Permeabilizes the cells with a higher Tween concentration and methanol
| A | B | |
| 870 μL | LICOR Intercept blocking buffer, PBS | |
| 50 μL | 20% (v/v) Tween 20 | |
| 80 μL | methanol | |
| 1 mL | total |
This buffer is made fresh each time
INTERCEPT blocking buffer, PBSLI-CORCatalog #927-70001
PermB buffer: Permeabilization buffer without methanol for washing cells and diluting antibodies
| A | B | |
| 950 μL | LICOR Intercept blocking buffer, PBS | |
| 50 μL | 20% (v/v) Tween 20 | |
| 1 mL | total |
This buffer is made fresh each time
PEM buffer : For washing cells of excess antibodies and matching the reflective index of water immersion objectives
| A | B | |
| 400 mL | 1M PIPES, ph 6.1 | |
| 5 mL | 1M EGTA | |
| 5 mL | 1M MgCl2 | |
| 1 L | total |
This can be made in bulk and stored at room temperature
Cell Fixation
Cell Fixation
10m
10m
Gently apply 50 µL PFA fix buffer and incubate for 00:05:00
Note
How to gently apply buffers henceforth.
1. Using gel loading pipette tips only, slowly pipette 50 µl of buffer on the left side of the well.
2. Afterwards, slowly remove 50 µl from the right side of the dish.
3. ALWAYS leave the cells covered in the remaining 50 µl of liquid.
4. All steps are performed at Room temperature .
5m
Gently apply 50 µL PFA/Tween fix buffer and incubate for 00:05:00
5m
Gently apply 50 µL glycine wash buffer and proceed immediately to permeabilization
Permeabilization and Blocking
Permeabilization and Blocking
1h
1h
Gently apply 50 µL PermB-meOH buffer and incubate for 00:15:00
15m
During the incubation, dilute antibodies in PermB buffer
| A | B | C | D | E | F | G | |
| Antigen | Source | Ex nm | Em nm | [Stock] | [Final] | Dilution | |
| alpha-Tubulin | Mouse (DM1A) | 0.5 mg/ml | 10 µg/ml | 1/500 | |||
| Rosetteless | Rabbit | UNK | UNK | 1/400 | |||
| Mouse IgG | Recombinant Nanobody | 568 | 603 | 1/500 | |||
| Rabbit IgG | Recombinant Nanobody | 647 | 665 | ||||
| DNA dye | POPO-1 | 434 | 456 | 0.1 mM | 0.1 µM | 1/1000 | |
| F-Actin | Phalloidin | 488 | 518 | 200 U/ml | 0.8 U/ml | 1/250 | |
| ALFA tag | Recombinant Nanobody | 650 | 670 | 5 µM | 20 nM | 1/250 |
Note
Henceforth keep the sample in the dark to protect from photobleaching!
Gently apply 50 µL PermB buffer
Note
Use PermB buffer WITHOUT antibodies, as this is only a wash step.
Apply Stains
Apply Stains
1h 10m
1h 10m
Gently apply 50 µL diluted antibodies . Repeat one more for a total of 2 applications to ensure the antibodies are not too dilute. Incubate for 01:00:00
Note
Cover the plate/samples during this incubation to minimize photobleaching.
1h
Gently apply 50 µL PEM . Repeat one more for a total of 2 applications to properly wash the cells of unbound antibodies.
Image Samples
Image Samples
10m
10m
With the cells in PEM buffer, proceed to imaging.
Protocol references
Götzke H, Kilisch M, Martínez-Carranza M, Sograte-Idrissi S, Rajavel A, Schlichthaerle T, Engels N, Jungmann R, Stenmark P, Opazo F, Frey S. The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications. Nat Commun. 2019 Sep 27;10(1):4403. doi: 10.1038/s41467-019-12301-7. PMID: 31562305; PMCID: PMC6764986.