Immunofluorescence (IF) Antibodies
A practical starting point for IF: choose targets, plan multiplex panels, and pick the right secondary antibodies & fluorophores. Includes organelle and lineage markers, signaling readouts (total vs phospho), and sample‑prep guidance.
Use indirect IF (primary + fluorescent secondary) for sensitivity and flexibility; use direct IF (fluorophore‑conjugated primary) for low background or multiplex with limited species. Match fixation/permeabilization to the epitope, and use cross‑adsorbed secondaries to avoid cross‑reactivity when multiplexing.
Organelle Markers
Classic organelle targets for IF; useful for co‑localization and compartment validation.
| Target (Gene) | Compartment | Notes | Browse |
|---|---|---|---|
| Lamin B1 (LMNB1) | Nuclear envelope | Outlines nuclei; good counterstain with DAPI. | Lamin B1 antibodies |
| Histone H3 (H3) | Chromatin | Nuclear marker; also used for PTM marks (H3K27me3). | H3 antibodies |
| Calnexin (CANX) | ER | Sheets around nucleus; reticular pattern. | Calnexin antibodies |
| PDI (P4HB) | ER | ER chaperone; bright perinuclear signal. | PDI antibodies |
| GM130 (GOLGA2) | Golgi | Juxtanuclear stacks; useful for polarity. | GM130 antibodies |
| TOM20 (TOMM20) | Mitochondria | Mito network; sensitive to fixation. | TOM20 antibodies |
| COX IV (COX4I1) | Mitochondria (IMM) | Inner membrane marker; punctate/filamentous. | COX IV antibodies |
| LAMP1 | Lysosome | Perinuclear puncta pattern. | LAMP1 antibodies |
| EEA1 | Early endosome | Puncta; overlaps partially with transferrin receptor. | EEA1 antibodies |
| Rab7A | Late endosome | Later endocytic vesicles; distinct from EEA1. | Rab7 antibodies |
| PEX14 | Peroxisome | Fine puncta; co‑stain with catalase (CAT). | PEX14 antibodies |
Cytoskeleton
Preserve architecture with appropriate fixation. Consider Phalloidin for F‑actin (probe) and antibodies for tubulins and intermediate filaments.
| Target (Gene) | Structure | Notes | Browse |
|---|---|---|---|
| α‑Tubulin (TUBA1A) | Microtubules | Filament network; better with methanol fixation. | α‑Tubulin antibodies |
| βIII‑Tubulin (TUBB3) | Neuronal MTs | Neuronal marker (TUJ1 clone). | βIII‑Tubulin antibodies |
| Vimentin (VIM) | Intermediate filaments | Mesenchymal cells; cytoplasmic cables. | Vimentin antibodies |
| Cytokeratin 18 (KRT18) | Intermediate filaments | Epithelial marker; pair with KRT8. | KRT18 antibodies |
Neuroscience
Core neuronal and glial IF markers for cultures and tissue.
| Target (Gene) | Cell type / Region | Notes | Browse |
|---|---|---|---|
| NeuN (RBFOX3) | Neurons (nuclei) | Nuclear/neuron‑specific; mature neurons. | NeuN antibodies |
| MAP2 | Neurons (dendrites) | Dendritic arbor; high MW. | MAP2 antibodies |
| GFAP | Astrocytes | Filamentous; upregulated in gliosis. | GFAP antibodies |
| PSD‑95 (DLG4) | Synapses (post‑synaptic) | Puncta along dendrites. | PSD‑95 antibodies |
| Synaptophysin (SYP) | Synapses (pre‑synaptic) | Vesicle puncta; co‑localize with PSD‑95. | Synaptophysin antibodies |
| MBP | Oligodendrocytes | Myelin sheaths; tissue sections. | MBP antibodies |
| TH (Tyrosine Hydroxylase) | Dopaminergic neurons | Neurotransmitter synthesis enzyme. | TH antibodies |
Immune Cell Markers
Surface markers and lineage proteins for immune profiling by IF; include Fc block in primary tissues.
| Target (Gene) | Cell type | Notes | Browse |
|---|---|---|---|
| CD45 (PTPRC) | Leukocytes | Pan‑leukocyte marker; membrane staining. | CD45 antibodies |
| CD3 (CD3E) | T cells | TCR complex component. | CD3 antibodies |
| CD4 | T helper | Membrane marker. | CD4 antibodies |
| CD8A | Cytotoxic T | Membrane marker. | CD8 antibodies |
| CD11b (ITGAM) | Myeloid | Membrane integrin. | CD11b antibodies |
| CD68 | Macrophages | Lysosomal protein; perinuclear puncta. | CD68 antibodies |
| Iba1 (AIF1) | Microglia | Cytoplasmic/filamentous in processes. | Iba1 antibodies |
| MHC class II (HLA‑DRA) | APCs | Membrane; species‑specific clones. | MHC II antibodies |
Epithelial / EMT
Track epithelial identity and EMT transitions in culture and tissue.
| Target (Gene) | Cell type / Compartment | Notes | Browse |
|---|---|---|---|
| E‑cadherin (CDH1) | Adherens junctions | Loss in EMT; membrane staining. | E‑cadherin antibodies |
| N‑cadherin (CDH2) | Adherens junctions | Gain in EMT; membrane staining. | N‑cadherin antibodies |
| Cytokeratin 8/18 (KRT8/18) | Intermediate filaments | Epithelial cytoskeleton. | KRT8/18 antibodies |
| Vimentin (VIM) | Intermediate filaments | Mesenchymal marker; EMT readout. | Vimentin antibodies |
| ZO‑1 (TJP1) | Tight junctions | Membrane junctions at apical surface. | ZO‑1 antibodies |
Endothelial
Vascular identity markers; useful in co‑culture and angiogenesis assays.
| Target (Gene) | Compartment | Notes | Browse |
|---|---|---|---|
| CD31 (PECAM1) | Membrane | Cell‑cell junctions; continuous lines. | CD31 antibodies |
| VE‑cadherin (CDH5) | Adherens junctions | Endothelial‑specific cadherin. | CDH5 antibodies |
| Von Willebrand Factor (VWF) | Secretory granules | Weibel‑Palade bodies; granular pattern. | VWF antibodies |
| Endomucin (EMCN) | Membrane | Microvascular endothelium marker. | Endomucin antibodies |
Cell Cycle & Proliferation
Combine nuclear and phospho markers to stage cells.
| Target (Gene) | Phase / Readout | Notes | Browse |
|---|---|---|---|
| Ki‑67 (MKI67) | Proliferation | Nuclear signal in cycling cells (G1–M). | Ki‑67 antibodies |
| p‑Histone H3 (Ser10) | Mitosis | Bright chromosomal signal in M phase. | pHH3 antibodies |
| Cyclin D1 (CCND1) | G1/S | Nuclear; mitogen responsive. | Cyclin D1 antibodies |
Apoptosis & Autophagy
Puncta and cleavage patterns are common IF readouts; validate with orthogonal assays where possible.
| Target (Gene) | Readout | Notes | Browse |
|---|---|---|---|
| Cleaved Caspase‑3 | Apoptosis | Cytoplasmic puncta/nuclear fragmentation in apoptotic cells. | Caspase‑3 antibodies |
| LC3B (MAP1LC3B) | Autophagosomes | Puncta increase with autophagy; co‑stain with p62. | LC3B antibodies |
| p62 / SQSTM1 | Autophagic flux | Decreases with active flux; puncta colocalize with LC3. | p62 antibodies |
| Beclin‑1 (BECN1) | Initiation | Perinuclear puncta; early autophagy. | Beclin‑1 antibodies |
Signaling (Total & Phospho)
Use phospho‑specific antibodies to capture activation patterns; keep cells on ice and include inhibitors to preserve PTMs.
| Target | Readout | Notes | Browse |
|---|---|---|---|
| p‑ERK1/2 (Thr202/Tyr204) | MAPK activity | Nuclear translocation after stimulation. | p‑ERK antibodies |
| p‑AKT (Ser473) | PI3K/AKT | Cytoplasmic/membrane signal; rapid kinetics. | p‑AKT antibodies |
| p‑STAT3 (Tyr705) | JAK‑STAT | Nuclear accumulation post‑cytokine. | p‑STAT3 antibodies |
| p‑AMPK (Thr172) | Energy stress | Cytoplasmic puncta/nuclear depending on cell type. | p‑AMPK antibodies |
Nuclear & Chromatin
Histone PTMs and transcription machinery—optimize permeabilization and include deacetylase inhibitors when staining for acetyl marks.
| Target | Readout | Notes | Browse |
|---|---|---|---|
| H3K27me3 | Repressive chromatin | Nuclear puncta/foci; cell‑type dependent. | H3K27me3 antibodies |
| H3K9ac | Active chromatin | Include HDAC inhibitors in buffers. | H3K9ac antibodies |
| RNA Pol II (Ser2P) | Transcription elongation | Nuclear speckles; distinct from Ser5P (initiation). | RNAPII Ser2P antibodies |
Stemness & Differentiation
Pluripotency triad and lineage markers for reprogramming and organoid work.
| Target (Gene) | Cell type / Readout | Notes | Browse |
|---|---|---|---|
| OCT4 (POU5F1) | Pluripotency | Nuclear; decreases on differentiation. | OCT4 antibodies |
| SOX2 | Pluripotency | Nuclear; co‑stain with OCT4/NANOG. | SOX2 antibodies |
| NANOG | Pluripotency | Nuclear puncta; early marker. | NANOG antibodies |
| Nestin (NES) | Neural progenitors | Intermediate filament; cytoplasmic fibers. | Nestin antibodies |
Recommended Secondary Antibodies for IF
Use highly cross‑adsorbed secondaries for multiplexing and to reduce species cross‑reactivity. Consider F(ab')2 fragments or Fab blocking in tissue to minimize Fc‑mediated background.
| Primary host | Recommended secondary | Common fluorophores | Notes | Browse |
|---|---|---|---|---|
| Rabbit | Anti‑Rabbit IgG (H+L) | AF488, AF555/568, AF594, AF647 | F(ab')2 to reduce background in tissue. | Rabbit IgG secondaries |
| Mouse | Anti‑Mouse IgG (H+L) | AF488, AF555/568, AF594, AF647 | For mouse tissue, use mouse‑on‑mouse or Fc‑specific options. | Mouse IgG secondaries |
| Rat | Anti‑Rat IgG (H+L) | AF488, AF555/568, AF594, AF647 | Good pairing with rabbit primaries in 2‑color IF. | Rat IgG secondaries |
| Goat | Anti‑Goat IgG (H+L) | AF488, AF555/568, AF594, AF647 | Use donkey to avoid goat‑on‑goat background. | Goat IgG secondaries |
| Chicken | Anti‑Chicken IgY (IgG) | AF488, AF555/568, AF594, AF647 | Low background with mammalian primaries. | Chicken IgY secondaries |
| Human | Anti‑Human IgG (H+L), Fcγ‑specific | AF488, AF555/568, AF594, AF647 | Use Fcγ‑specific to reduce Fc receptor binding. | Human IgG secondaries |
Fluorophore Selection & Spectral Planning
Choose spectrally separated dyes with similar brightness and photostability. For 3–4 colors, pair 405/488/555/647 channels and keep the brightest dye for the weakest target.
| Fluorophore | Excitation / Emission (nm) | Typical laser | Notes | Browse |
|---|---|---|---|---|
| Alexa Fluor 488 | 495 / 519 | 488 nm | Bright green channel; low bleed into 555. | AF488 secondaries |
| Alexa Fluor 555 / 568 | 555–578 / 565–603 | 561 nm | Orange/red; pair with AF488 and AF647. | AF555/568 secondaries |
| Alexa Fluor 594 | 590 / 617 | 561 nm | Deeper red; can overlap with 568—avoid concurrent use. | AF594 secondaries |
| Alexa Fluor 647 | 650 / 668 | 633–640 nm | Far‑red; excellent for low abundance targets. | AF647 secondaries |
Fixation & Permeabilization
Match fixation to epitope: methanol preserves microtubules and phospho‑epitopes; PFA preserves structure but may require antigen retrieval or stronger permeabilization.
| Target type | Recommended fixation | Permeabilization | Notes |
|---|---|---|---|
| Microtubules / Tubulin | −20°C methanol (5–10 min) | None or mild (0.05% Triton) | Methanol stabilizes MTs; avoid over‑extraction. |
| Actin / Vimentin / IFs | 4% PFA (10–15 min) | 0.1–0.3% Triton X‑100 | Preserve architecture; Phalloidin for F‑actin (probe). |
| Membrane proteins | 4% PFA | 0.1% Saponin | Saponin permeabilizes cholesterol‑rich membranes gently. |
| Nuclear proteins / Histones | 4% PFA | 0.2–0.5% Triton X‑100 | Consider acid extraction for histone marks on WB; IF needs strong permeabilization. |
| Phospho‑epitopes | −20°C methanol or PFA + inhibitors | 0.1% Triton X‑100 | Include phosphatase inhibitors during fixation and washes. |
Controls & Counterstains
Always include negative controls and a nuclear counterstain; for tissue, consider autofluorescence quenching and Fc block.
| Control | Purpose | Notes | Browse |
|---|---|---|---|
| No primary control | Detects non‑specific secondary binding | Signal here indicates secondary or tissue autofluorescence. | Secondaries |
| Isotype control | Matches primary isotype | Mouse IgG1, IgG2a; Rabbit IgG, etc. | Isotype controls |
| Fc block | Reduce Fc receptor binding | Especially in immune tissues (spleen, blood). | |
| DAPI / Hoechst | Nuclear counterstain | Mount with anti‑fade medium. |
IF FAQs
Quick answers to common IF questions.
| Question | Answer |
|---|---|
| Direct vs indirect IF — which should I use? | Indirect IF (primary + labeled secondary) offers higher sensitivity and panel flexibility. Direct IF (labeled primary) reduces background and is handy when species are limited. |
| How do I reduce background? | Titrate antibodies, increase blocking (serum/BSA), include Fc block for immune tissues, use cross‑adsorbed secondaries, shorten exposure, and consider F(ab')2 fragments. |
| How do I plan a 4‑color panel? | Pick spectrally separated dyes (405/488/555/647), assign the brightest to the weakest target, and avoid using both 568 and 594 together. |
Need help building an IF panel?
Tell us your species, targets, microscope channels, and fixation method — we’ll suggest primaries and cross‑adsorbed secondaries that fit.