Microscopy & Imaging Protocols — Armin Bayati, PhD

01

IMMUNOFLUORESCENCE

Immunofluorescence (IF) Protocol

Bench-ready fluorescence staining for cultured cells using immunofluorescent antibodies. Optimized for confocal microscopy with sequential scanning to minimize crosstalk. Yields high signal-to-noise ratios for accurate colocalization analysis.

Leica TCS SP8

Confocal

DAPI

Counterstain

Dako Medium

Mounting

63× Oil

Objective

PHASE 1 Fixation & Permeabilization (~45 min)

▼

1

Cell Fixation

Fix with 2% PFA in PBS for 10–15 min on ice. Aspirate and wash 2× with cold PBS.

2% PFAPBS

⏲ 15 min

2

Blocking & Permeabilization

0.05% Triton X-100 + 5% BSA in PBS for 30 min at RT.

Triton X-100BSAPBS

⏲ 30 min

PHASE 2 Antibody Staining (3–4h)

▼

3

Primary Antibody

1:500 in 0.01% Triton/5% BSA, 2h RT in humidified chamber.

Primary AbDilution Buffer

⏲ 2h

4

Wash

3× PBS, 5 min each.

PBS

⏲ 15 min

5

Secondary Antibody

1:500 fluorophore-conjugated, 1h RT protected from light.

Secondary Ab-Fluorophore

⏲ 1h

6

Wash & Counterstain

2× PBS, DAPI 1 µg/mL 10 min RT, 1× PBS.

DAPIPBS

⏲ 15 min

PHASE 3 Mounting & Imaging

▼

7

Mounting

Dako Fluorescence Mounting Medium, cure overnight 4°C dark.

Dako Medium

⏲ Overnight

8

Confocal Imaging

Leica TCS SP8, 63× oil, sequential scanning, analyze with LAS X 3.5.5.

Leica TCS SP8

⏲ Variable

02

IMMUNOHISTOCHEMISTRY

Immunohistochemistry (IHC) Staining Protocol

Standard chromogen-based IHC for paraffin sections with DAB detection. Includes antigen retrieval, blocking, and streamlined antibody staining with horseradish peroxidase (HRP) detection.

Microtome

Sectioning

Citrate pH 6.0

HIER

DAB Substrate

Chromogen

Hematoxylin

Counterstain

PHASE 1 Tissue Preparation (~1h)

▼

1

Sectioning

5–7 µm paraffin on rotary microtome, float in 56°C water bath, mount on charged slides, bake 50–60°C 20 min.

Microtome

⏲ 30 min

2

Deparaffinization & Rehydration

Xylene 2×5 min → 100% EtOH 2×3 min → 95% 3 min → 70% 3 min → 50% 3 min → dH₂O.

XyleneGraded EtOH

⏲ 25 min

PHASE 2 Antigen Retrieval & Blocking (~1.5h)

▼

3

Endogenous Peroxidase Block

3% H₂O₂ in methanol, 10 min RT, wash 3× PBS.

3% H₂O₂Methanol

⏲ 15 min

4

HIER

10 mM citrate buffer pH 6.0, 95–100°C 10 min, cool 20 min RT, wash 2× PBS.

Citrate Buffer pH 6.0

⏲ 35 min

5

Serum Blocking

10% normal serum in PBS, 1h RT humidified.

Normal SerumPBS

⏲ 1h

PHASE 3 Antibody & Detection (Overnight + 2h)

▼

6

Primary Antibody

Per manufacturer, overnight 2–8°C, include negative control.

Primary Ab

⏲ Overnight

7

Secondary Antibody

Wash 3× PBS, biotinylated secondary 1:200–1:500, 30–60 min RT, wash 3× PBS.

Biotinylated Secondary

⏲ 45 min

8

ABC Complex

Streptavidin-HRP 30 min RT, wash 3× PBS.

Streptavidin-HRP

⏲ 30 min

PHASE 4 Chromogen & Counterstain (30 min)

▼

9

DAB Development

DAB substrate (0.05% DAB + 0.015% H₂O₂ in PBS), monitor <5 min, stop in dH₂O. Caution: DAB is suspected carcinogen.

DAB Substrate

⏲ <5 min

10

Counterstain & Mount

Hematoxylin 0.5–5 min, rinse, blue in Scott's water, dehydrate, clear xylene, mount permanent.

HematoxylinXyleneDPX

⏲ 20 min

03

STED MICROSCOPY

STED Super-Resolution Protocol

Stimulated emission depletion (STED) nanoscopy achieves sub-diffraction resolution (<50 nm) for ultrastructure imaging. Uses 775 nm depletion beam with compatible fluorophores and optimized acquisition parameters.

Abberior STED

Nanoscope

775 nm

Depletion

100× NA 1.4

Objective

<50 nm

Resolution

PHASE 1 Sample Preparation (~4h)

▼

1

Standard IF Protocol

Follow Section 01 through fixation, blocking, antibody staining. Use STED-compatible fluorophores (Abberior STAR 580, STAR 635P, ATTO 647N).

STED-Compatible Secondaries

⏲ ~4h

2

STED Mounting

ProLong Diamond or Abberior Mount Liquid. Use #1.5H precision coverslips (170 ± 5 µm). Cure completely.

ProLong Diamond#1.5H Coverslips

⏲ Overnight

PHASE 2 STED Imaging

▼

3

Instrument Setup

Align 775 nm depletion beam. Calibrate with 40 nm crimson beads. Optimize power (start low).

Abberior STED

⏲ Variable

4

Acquisition

100× oil NA 1.4, pixel size 15–20 nm (Nyquist), line accumulation 2–4×, acquire confocal reference first then STED.

⏲ Variable

5

Image Processing

Deconvolve with Huygens or Richardson-Lucy. Analyze in ImageJ/FIJI. Measure FWHM of point-like structures.

HuygensImageJ/FIJI

⏲ Variable

04

IN VITRO TEM

In Vitro Transmission Electron Microscopy (TEM) Protocol

Cultured cell TEM sample preparation using chemical fixation, osmium post-fixation, en bloc uranyl staining, and Spurr's resin embedding. Yields ultrathin sections for ultrastructural analysis at 70–80 nm thickness.

Tecnai Spirit 120 kV

In Vitro TEM

Gatan Ultrascan 4K

Camera

OsO₄

Post-fixation

Uranyl Acetate

Contrast

PHASE 1 Fixation & Post-fixation (~3h)

▼

1

Primary Fixation

2.5% glutaraldehyde in 0.1 M sodium cacodylate on Permanox plates, 1–2h RT or overnight 4°C.

2.5% GA0.1 M Cacodylate

⏲ 1–2h

2

Post-fixation

Wash 3× cacodylate buffer. 1% OsO₄ + 1.5% potassium ferrocyanide in cacodylate, 1h RT. Wash 3×. Caution: OsO₄ highly toxic—fume hood.

1% OsO₄K₄[Fe(CN)₆]

⏲ 1h

3

En Bloc Staining

4% aqueous uranyl acetate, 1h RT or overnight 4°C. Wash 3× dH₂O.

4% Uranyl Acetate

⏲ 1h

PHASE 2 Dehydration & Embedding (~2 days)

▼

4

Dehydration

Graded ethanol: 50%, 70%, 80%, 95% (15 min each), 100% (2× 30 min).

Graded Ethanol

⏲ 2.5h

5

Resin Infiltration

1:1 ethanol:Spurr's 1h → pure Spurr's overnight on rotator → fresh Spurr's 2–4h.

Spurr's Resin

⏲ Overnight + 4h

6

Embedding & Polymerization

BEEM capsules or flat molds, polymerize 60°C 48h.

BEEM Capsules

⏲ 48h

PHASE 3 Sectioning & Imaging

▼

7

Ultrathin Sectioning

Trim with glass knives, cut 70–80 nm with diamond knife (Diatome Ultra 35°) on ultramicrotome, collect on 200-mesh Cu grids.

UltramicrotomeDiamond Knife

⏲ Variable

8

Grid Staining (Optional)

Uranyl acetate 5 min + lead citrate 2 min if no en bloc staining.

UAReynold's Lead Citrate

⏲ Variable

9

TEM Imaging

Tecnai G2 Spirit 120 kV, Gatan Ultrascan 4000 CCD (4008×2672 px), 5,000× to 80,000×.

Tecnai SpiritGatan Camera

⏲ Variable

05

IN VIVO TEM

In Vivo Transmission Electron Microscopy (TEM) Protocol

Whole-animal perfusion-fixation followed by tissue dissection, vibratome sectioning, and TEM sample preparation. Preserves native ultrastructure in tissue and organs with minimal artifact.

JEM-1400 JEOL

In Vivo TEM

Leica VT1000S

Vibratome

Spurr's Resin

Embedding

Perfusion

Fixation

PHASE 1 Perfusion Fixation (~30 min)

▼

1

Anaesthesia & Perfusion Setup

Deep anaesthesia per IACUC, thoracotomy, cannulate left ventricle, nick right atrium.

Anaesthetic

⏲ 10 min

2

PBS Flush

Heparinized PBS (37°C) at steady rate 5 min, verify blanching.

Heparinized PBS

⏲ 5 min

3

Fixative Perfusion

2% glutaraldehyde + 2% PFA in 0.15 M cacodylate, ~75 mL slowly over 10 min, observe fixation rigor.

2% GA/2% PFA

⏲ 10 min

PHASE 2 Tissue Processing (~24h + 2 days)

▼

4

Dissection & Post-fixation

Dissect target organs, immerse in fixative 24h at 4°C.

Fixative

⏲ 24h

5

Vibratome Sectioning

200 µm on Leica VT1000S, microdissect ROI under stereomicroscope (Olympus SZ61).

Leica VT1000S

⏲ 1–2h

6

OsFeCN Staining

1% osmium tetroxide-potassium ferrocyanide in cacodylate, 2h RT on rotator, wash 3×.

1% OsFeCN

⏲ 2h

7

En Bloc Uranyl Acetate

2% uranyl acetate 2h on rotator, wash 3× dH₂O.

2% UA

⏲ 2h

PHASE 3 Dehydration & Embedding (~3 days)

▼

8

Dehydration

50%, 70%, 80%, 95% (15 min each), 100% (2× 30 min).

Graded Ethanol

⏲ 2.5h

9

Resin Infiltration

1:1 ethanol:Spurr's 1h → pure Spurr's overnight on rotator.

Spurr's Resin

⏲ Overnight

10

Embedding

Orient tissue in BEEM capsules, polymerize 60°C 48–72h.

BEEM Capsules

⏲ 48–72h

PHASE 4 Sectioning & TEM Imaging

▼

11

Semi-thin Sectioning

~0.5 µm with glass knives on Reichert-Jung UltraCut E, stain toluidine blue for light microscopy survey.

Ultramicrotome

⏲ Variable

12

Ultrathin Sectioning

70–80 nm with diamond knife (Diatome Ultra 35°), collect on 200-hex Cu mesh grids. Verify thickness by interference color (silver/gold).

Diamond Knife

⏲ Variable

13

TEM Imaging & Analysis

JEM-1400 (JEOL) at 80–120 kV, Orius SC1000 camera (Gatan, 4008×2672 px). Quantitative analysis at 30,000× (test field 6.58 × 4.39 µm = 28.89 µm²).

JEM-1400Orius Camera

⏲ Variable

06

EQUIPMENT

Equipment & Software Summary

Complete inventory of instrumentation and analysis tools supporting all microscopy protocols described on this page.

Leica TCS SP8

Confocal Microscopy

Abberior STED

Super-Resolution

Tecnai Spirit

120 kV · In Vitro TEM

JEM-1400

JEOL · In Vivo TEM

Leica VT1000S

Vibratome

ImageJ / FIJI

Image Analysis

LAS X 3.5.5

Leica Software

GMS 3

Gatan Software