This video describes the immunohistochemical staining technique to identify dendritic cells from endobronchial biopsies.
Protocol
1. GMA sectioning. Prepare 0.05% ammonia solution with distilled water. Place the glass knife and biopsy in the microtome. Carefully align the biopsy block with the blade by adjusting the knife holder and the angle of the biopsy block so that the blade rests flat against the surface of the block. Cut 2 µm thick sections using the microtome on a slow cutting speed and use forceps to transfer and float out sections on the ammonia water. Float the sections for 45-90 sec, allowing gentle antigen re…
Declarações
The authors have nothing to disclose.
Materials
GMA sectioning
Glass microscope slides
ThermoFisher Scientific
10143562CEF
Cut edges, frosted end
Poly-L-Lysine solution
Sigma-Aldrich
P8920-500mL
1:10 for working solution
Sheet glass strips for ultramicrotomy
Alkar
Tween 20
Sigma-Aldrich
P2287
Wash solution (0.1% Tween20)
LKB 7800B Knifemaker
LKB
Capsule splitter
TAAB laboratories
C065
Carbon steel single edge blades
TAAB laboratories
B054
Vice
Ammonia, 25%
VWR
1133.1
2 ml in 1 L, 1:500 (0.05%)
Microtome
Leica
Leica RM 2165
Light source
Leica
Leica CLS 150 XE
Microscope with swing arm stand
Leica
Leica MZ6
GMA Immunohistochemistry
Diamond tipped pen
Histolab
5218
Hydrogen peroxide 30% solution
AnalaR Normapur
23619.264
Sodium azide
Sigma-Aldrich
S8032
Tris
Roche
10708976001
Sodium chloride
VWR chemicals
27810.295
Bovine serum albumin
Millipore
82-045-2
Probumin BSA diagnostic grade
Dulbecco's modified eagle medium
(DMEM)
Sigma-Aldrich
D5546
Anti-human CD45 antibody
BioLegend
304002
Mouse monoclonal, clone HI30, isotype IgG1k. Working concentration of 500 ng/ml
Anti-human CD1a antibody AbD
Serotech
MCA80GA
Mouse monoclonal, clone NA1/34- HLK, isotype IgG2a. Working concentration of 10 µg/ml
Immunohistochemical Staining: A Method for Obtaining Spatial Information from Thin Tissue Sections. J. Vis. Exp. (Pending Publication), e20247, doi: (2023).