Feb 24, 2022
Immunofluorescent staining of formalin-fixed OCT-embedded human lung tissue
- Justinn Barr1,
- Jamie Verheyden1,
- Xin Sun1,2
- 1Department of Pediatrics, University of California, San Diego, CA 92093, USA;
- 2Division of Biological Sciences, University of California, San Diego, CA 92093, USA
- SPARCTech. support email: info@neuinfo.org
Protocol Citation: Justinn Barr, Jamie Verheyden, Xin Sun 2022. Immunofluorescent staining of formalin-fixed OCT-embedded human lung tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.b2jnqcme
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: December 02, 2021
Last Modified: February 24, 2022
Protocol Integer ID: 55630
Abstract
This protocol describes steps for immunofluorescent staining of formalin-fixed paraffin-embedded human lung tissue.
Materials
Primary Antibodies
| A | B | C | D | E | F | |
| Antibody | Dilution | Company | Catalog Number | Lot number | RRID | |
| Neuron-specific beta-III Tubulin Biotin MAb (Clone TuJ-1) | 1:100 | R&D Systems | BAM1195 | HVS0215121 | AB_356859 | |
| Human CD31/PECAM-1 MAb (Clone 9G11) | 1:100 | R&D Systems | BBA7 | AB_356960 | ||
| Anti-Tyrosine Hydroxylase Antibody | 1:100 | MilliporeSigma | AB152 | AB_390204 | ||
| Anti-Synaptophysin (Neuroendocrine Marker) Ab-4 Polyclonal Antibody, Unconjugated | 1:100 | Fisher Scientific | RB-1461-P0 | 1461P1603G | AB_60082 | |
| Monoclonal Anti-Actin, alpha-Smooth Muscle - Cy3 | 1:100 | Sigma-Aldrich | C6198 | 086M4829V | AB_476856 | |
| Anti-VAChT (rabbit, polyclonal) | 1:100 | Synaptic Systems | 139 103 | AB_887864 | ||
| Anti-TAGLN/Transgelin (SM22) | 1:100 | Abcam | ab14106 | GR274712-1 | AB_443021 | |
| Anti-Calcitonin Gene Related Peptide (CGRP) antibody produced in rabbit | 1:100 | Sigma-Aldrich | C8198 | 066M4836V | AB_259091 | |
| Anti-P2X3 Receptor | 1:100 | Sigma-Aldrich | AB5895 | AB_177544 | ||
| Anti-VGLUT 2 antibody | 1:100 | Synaptic Systems | 135 403 | AB_887883 | ||
| Anti-PGP9.5 [13C4 / I3C4] | 1:100 | Abcam | ab8189 | AB_306343 |
Primary Antibodies
Secondary Antibodies
| A | B | C | D | E | F | |
| Antibody | Dilution | Company | Catalog Number | Lot number | RRID | |
| Cy3-AffiniPure Goat Anti-Mouse IgG | 1:300 | Jackson ImmunoResearch Labs | 115-165-003 | 129939 | AB_2338680 | |
| Fluorescein (FITC)-AffiniPure Goat Anti-Rabbit IgG (H+L) | 1:300 | Jackson ImmunoResearch Labs | 111-095-144 | AB_2337978 |
Secondary Antibodies
2X5 min wash in Phosphate-buffered saline with Tween (PBSTW) in a slide holder on a flat rotator.
Blocking:
Mix up 5% serum (50 µL serum / 1 mL of PBSTW)
Note
Normal goat serum in most cases.
Remove old PBSTW from the sample and add block.
Cover with Hybrislip.
Let sit in a humidified chamber at Room temperature for ≥ 1 hour.
Primary antibody:
Mix up more of the 5% serum.
Add antibody to the serum at the desired concentration.
Take the humidified chamber to a cold room.
Add primary antibody in the cold room.
Cover with Hybrislip.
Seal the humidified chamber.
On Day 2:
Wash slides 3X10 min at Room temperature in slide holder on a flat rotator.
Secondary antibody:
Mix up 5% serum.
Add secondary antibody to the serum at the desired concentration.
Add antibody mix to the slides.
Let it sit in a humidified chamber EITHER at Room temperature for 1 hour OR in the cold room overnight.
Cover with Hybrislip.
Wash:
1x10 min in PBSTW with DAPI.
2x10 min in PBSTW.
Mount the slides in VectaShield and seal with nail polish.