Immunostaining& data analysis
Shujing LiION, SIBS, CAS20141127
Immunostaining• Detect specific molecules in cells / tissues
– proteins, peptides, sugars, lipids, etc.
• based on antibody‐antigen recognition– Specificity– Sensitivity
WHY do immunostaining?• Where
Spatial location – e.g. specific brain nuclei, subcellular localization
• When
Cellular events – e.g. apoptosis
• Cell type‐specific biomarkers
e.g. CD4, CD8 T‐cells, NK, Macrophages, Mast Cells, etc.
• Levels or efficiency of expression
Compare immunostaining, WB vs ELISA
Immunostaining WB ELISA
Sensitivity
Specificity
Quantitative
Localization
Immunostaining workflowIHC: Immunohistochemisry
Blocking
1st Ab
2nd Ab
Staining with substrates Mounting
Antigen retrievalPermeabilization
FixationEmbedding
ICC: Immunocytochemistry
IF: Immunofluorescence
Immuno‐elelctron microscopy
FixationGoals of fixation
1. Prevent autolysis by rapidly terminating
enzymatic/metabolic activities
2. Preserve tissue structures while stabilizing and hardening the tissue for processing.
3. Prevent bacterial decomposition
Key factors affecting chemical fixation1. Osmolality, pH, temperature 2. Concentration of fixative3. Incubation time4. Tissue type5. Size of specimen: No more than 4mm thick(fixative penetration)
A: 1 hour (approximately 0.8 mm penetration), B: 2 hours (approximately 1.2 mm penetration), C: 4 hours (approximately 1.6 mm penetration) and D: 8 hours (approximately 2.2 mm penetration)
Fixatives‐Aldehydes(formaldehyde 甲醛, glutaraldehyde戊二醛)‐Oxidizing agents, metallic ions and complexes(osmium tetroxide 四氧化锇, chromic acid 铬酸, Zn)‐Protein‐denaturing agents(acetic acid 乙酸, methanol 甲醇, ethanol 乙醇, acetone 丙酮)
氯化汞,重铬酸钾
Effect of fixative on immunostaining patterns
http://www.leicabiosystems.com/pathologyleaders/fixation‐and‐fixatives‐4‐popular‐fixative‐solutions/
EmbeddingParaffin (wax) sections Cryostat (frozen) sections
Storage Multiple years at room temperature 1 year at ‐80
Morphology Preserve tissue morphology and subcellular structure well (but brain & spinal cord may shrink)
Ice crystals may affect tissue structure
Antigen preservation Antigen masking, often need antigen retrieval
Preserve antigen (but also retain enzyme activity)
Protocols Multiple proceduredehydrate & rehydrate
Time‐saving
Antigen retrieval• Proteolytic‐Induced Epitope Retrieval (PIER): Enzymatic digestion
• Heat‐Induced Epitope Retrieval (HIER): Microwave, Pressure cooking
Emoto et al., J Histochem Cytochem, 2005
Effects of pH on heat‐induced antigen retrieval
Permeabilization(if detecting an intracellular target)
Sang Hyoung Lee,…Morgan Sheng, Neuron, 2004
Blocking• Blocking non‐specific binding of antibodies
‐BSA or a serum matching the species of 2nd Ab• Biotin blocking: avidin and biotin • Peroxidase blocking: H2O2 • Alkaline phosphatase blocking: levisamole• Autofluorescence blocking:
‐Glycine/lysine or sodium borohydride to block aldehydes‐Use frozen tissue sections
• Blocking cross‐reactive antigens (Mouse on Mouse, 2nd Ab binding to endogenous mouse IgG and to Fc receptors)‐Unconjugated anti‐mouse IgG (H+L)‐Using F(ab) monomeric secondary antibodies
Primary antibodiesPolyclonal – a mixture of multiple antibodies recognizing different epitopes of the antigen
Monoclonal – identical copies of the same unique antibody. Recognizes only a single epitope
HOW to prove an antibody is specific and efficient? Negative control: no 1st Ab, KO or knock‐down samples, region‐specific pattern Positive control: overexpression samples, region‐specific pattern Antigen absorptionOther evidence: WB etc.
No signalIf positive control not work: protocol, solution, tissue/cell sample, 2nd AbIf positive control work: 1st Ab concentration, ABC or TSA amplification, antigen retrieval
High backgroundIf negative control also have signal/background: blocking, dry, staining with substrates too longIf negative control have no signal/background: blocking, 1st Ab concentration
Secondary antibodies & substratesFluorescence dyes: ALEXA dyes, Rhodamine, Cy5, etc.HRP (horseradish peroxidase), DABAP (Alkaline phosphatase), NBT/BCIP
Polymer method
TSA: Tyramide Signal Amplification
Mounting• Fluorescent imaging
Aqueous mounting media (hydrophilic), anti‐fading, glycerol
• Enzymatic label / chromogen combinationsOrganic mounting media (hydrophobic)
Substrates and chromogens for IHC
Lasers & Fluorescent dyeslasers
Dye Ex Peak(nm)
Em Peak(nm)
DAPI (nucleus) 358 461GFP 470 509
Alexa 488 495 519FITC 495 519
Propidium Iodide (nucleus) 536 617Alexa 568 578 603
TRITC 547 572Cy3 550 570
Texas Red 595 615Alexa 633 632 647
TOTO-3, TOPRO-3 (Nucleus) 642 660Cy5 650 670
500
600
700
488
543
633
405Immunofluorescence• Advantages– Hi‐resolution, easy to double/triple label– Better subcellular detail– Can be used with 3D microscopy/live imaging• Disadvantages– Background autofluorescence– Lack of surrounding tissue/cellular detail– Not permanent
Commonly used fluorescent molecules• Nuclear stains: DAPI, PI, TOPRO, TOTO…• Calcium indicators: Fura, Fluo• Membrane dyes: DiI, DiO• Synaptic vesicle labels: FM1‐43, FM4‐64• Organelle marker: MitoTracker• Cytoskeleton marker: Phalloidin (F‐actin)
Color vs Brightness
Image quantitative analysisIntensity information from immunostaining images are RELATIVE (not absolute) and NON‐LINEARQuantitative analysis only work on high‐quality images
Before start…Experiment design• Always include a CONTROL in each experiment: eg. GFP transfection, neighboring untransfected neuron, untreated neuronal culture, vehicle (DMSO, EtOH), depending on your sample and your hypothesis
• Co‐process all samples simultaneously: data is relative and not absolute
• Make master mixes for antibodies when possible
• Multiple samples of each treatment per experiment, repeated in multiple experiments (at least 3, but COMPLETE one full experiment before repeating: your experiment design usually needs to be retuned unless you are a GENIUS)
• Keep all acquisition parameters constant (Laser intensity, Detector gain, Objective, Light path, Zoom, Frame size, Averaging / Scanning speed, Pinhole size, Projection, number of layers)
• Collect images on same day if possible (fading problems, exact same microscope settings etc)
• Beginners: re‐check parameters of images throughout experiment even if you did not actively change anything
• Blind‐code all data on acquisition (good habit, very important for qualitative analyses)
• Always keep unprocessed original image (data can be reanalyzed years later, as long as you have the original data with all the controls)
Collecting images for quantitative analysis
Data analysis
• Number (cell number, branch tip number, spine number)• Length (dendrite length, spine neck & diameter)• Area • Intensity • Co‐localization
Example 1: what’s the density of spine? Number/length
Example 2: How does activity affect the expression of TF in neurons?
neuron markerAlexa 568
TFAlexa 488
Application in neurosciencesFluorescence activated cell sorter (FACS) Immuno‐electron microscopy
Application in neurosciencesFluorescence in situ hybridization (FISH)
Jianbo Xiu,…Hailan Hu, Nat Neurosci., 2014
Tyramide‐amplified IHC–FISH (TAI‐FISH)