CHAPTER THIRTEEN
Immunohistochemistry on Freely Floating Fixed Tissue Sections Julia Bachman1 Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Theory 2. Equipment 3. Materials 3.1 Solutions & buffers 4. Protocol 4.1 Preparation 4.2 Duration 5. Step 1 Pretreatment of Tissue Section 5.1 Overview 5.2 Duration 6. Step 2 Blocking Nonspecific Binding 6.1 Overview 6.2 Duration 7. Step 3 Primary Antibody Labeling 7.1 Overview 7.2 Duration 7.3 Tip 8. Step 4 Secondary Antibody Labeling 8.1 Overview 8.2 Duration 9. Step 5 Mount Labeled Tissue Section on Microscope Slide 9.1 Overview 9.2 Duration 9.3 Tip 9.4 Tip
208 208 208 209 210 210 210 210 210 210 211 211 211 212 212 212 213 213 213 213 214 214 214 215 215
Abstract Immunohistochemistry on free floating tissue sections is done for many reasons, all of which involve labeling tissue to visualize a certain cell type, protein, or structural component. Visualization is aided by mounting sections on microscope slides for stabilization, and is in most cases necessary for the appropriate use of objectives with a high numerical aperture and high degree of magnification. Methods in Enzymology, Volume 533 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-420067-8.00013-1
#
2013 Elsevier Inc. All rights reserved.
207
208
Julia Bachman
1. THEORY In order to visualize a given component of a tissue sample, the tissue is fixed, sectioned, and labeled. Fixation preserves the molecular organization of the tissue by cross-linking proteins. The most common method of fixation uses 4% paraformaldehyde. Fixation can be done before or after sectioning, depending on the experimental design. Sectioning tissue allows for a more detailed analysis, since thinner sections are more easily penetrated by labeling agents (antibodies and dyes) and by light. Detergent is then used to permeabilize the tissue, which enhances label penetration. The following protocol is for immunolabeling tissue sections – using antibody-based identification to probe particular molecules. The sections are blocked with high amounts of serum to reduce nonspecific binding of subsequent antibody treatments. Primary antibodies specifically label a protein of interest (POI), while secondary antibodies are used for signal amplification and are often conjugated to a visualization signal, such as a fluorophore.
2. EQUIPMENT Flame-polished Pasteur pipette (in hook-shape, for transferring sections) Note: Plastic Pasteur pipettes can also be used to transfer delicate or small tissue (just make sure that the opening is wider than the specimen size and work slowly, so as not to introduce air bubbles into the tissue). Rotator Microscope slides and coverslips (adjust both to size of tissue (length and width) and working distance of microscope (thickness)) Fine paintbrush Glass or hard plastic dish or container with inserts with mesh bottoms (for washing samples, inserts allow for easy transfer of sections between washes) Note: If specimens are small enough (e.g., Drosophila salivary glands), one can do all incubations and washes in a microcentrifuge tube. Change solutions by either pipetting or vacuum aspiration. Aluminum foil
3. MATERIALS Primary antibody (to protein of interest) Appropriate secondary antibody conjugated to a fluorophore Sodium chloride (NaCl)
209
Immunohistochemistry on Freely Floating Fixed Tissue Sections
Potassium chloride (KCl) Sodium phosphate dibasic (Na2HPO4) Potassium phosphate monobasic (KH2PO4) Triton X-100 Normal Goat Serum (NGS. Alternatives: Normal Donkey Serum, Bovine Serum Albumin) Tissue section mounting media (large variety, all commercially available) Nail polish
3.1. Solutions & buffers 10 PBS Component
Final Concentration
Amount
NaCl
136.9 mM
100 g
KCl
2.68 mM
2.5 g
Na2HPO4
10.1 mM
18 g
KH2PO4
1.76 mM
3g
Dissolve the components in 800 ml purified water. Adjust the pH to 7.5 with NaOH. Add purified water to 1 l
PBST Component
Final Concentration
Stock
Amount
PBS
1
10
50 ml
Triton-X-100
0.3%
10%
15 ml
Add to 435 ml purified water
Blocking Buffer Component
Final Concentration
Stock
Amount
NGS
10%
100%
1 ml
PBST
1
1
9 ml
Component
Final Concentration
Stock
Amount
NGS
2%
100%
200 ml
PBST
1
1
9.8 ml
Incubation Buffer
210
Julia Bachman
4. PROTOCOL 4.1. Preparation Tissue fixation varies depending on the desired component to be visualized, tissue type, and tissue thickness. For example, rodents are typically perfused first with saline, followed by fixative (4% paraformaldehyde). The region or organ of interest is dissected out and incubated in cold fixative for an additional 20 min to 2 h. The tissue is then incubated in increasing amounts of sucrose (10%, 20%, and 30%) for several hours or overnight each. Sucrose serves as an isotonic solution that will prevent tissue shrinkage. The tissue is then sectioned using a microtome or vibratome. Fixation after sectioning requires only a brief (10–20 min) incubation in fixative. Use the flame-polished, hooked Pasteur pipette to transfer sections from wash containers to tubes already containing the appropriate solution. See Video 1. http://dx.doi.org/10.1016/B978-0-12-420067-8.00013-1. Make sure to rinse the pipette if transferring sections to multiple tubes containing different antibodies. When transferring sections from tubes back into wash containers, dump contents into container and use a plastic pipette to rinse the tube, making sure all sections have been removed.
4.2. Duration Preparation
Variable
Protocol
2 h (day 1) 3.5 h (day 2)
See Fig. 13.1 for the flowchart of the complete protocol.
5. STEP 1 PRETREATMENT OF TISSUE SECTION 5.1. Overview Tissue is permeabilized by a light treatment of detergent. This allows for better penetration of antibodies and buffers into the tissue.
5.2. Duration 35 min 1.1 Quickly rinse tissue sections in PBST.
Immunohistochemistry on Freely Floating Fixed Tissue Sections
211
Figure 13.1 Flowchart of the complete protocol, including preparation.
1.2 Incubate in fresh PBST for 10–15 min. 1.3 Wash 4 times in PBS – 5 min each wash. See Fig. 13.2 for the flowchart of Steps 1 and 2.
6. STEP 2 BLOCKING NONSPECIFIC BINDING 6.1. Overview Incubating the sample with a high concentration of serum decreases non specific binding of the antibody to the tissue. Other antibodies and components of the serum will bind to ‘stickier’ areas of the tissue thus blocking binding of the antibody used for the specific stain in question.
6.2. Duration About 1 h 2.1 Fill 1.5-ml round bottom microcentrifuge tubes with 1 ml of Blocking Buffer. 2.2 Transfer the tissue sections into tubes.
212
Julia Bachman
Figure 13.2 Flowchart of Steps 1 and 2.
2.3 Rotate at room temperature for 1 h. In the meantime, prepare solutions for incubation with primary antibody. 2.4 Quickly rinse sections in PBS.
7. STEP 3 PRIMARY ANTIBODY LABELING 7.1. Overview Tissue is incubated with antibody raised against specific proteins of interest. The serum concentration is lowered to aid specific binding of the antibody. Any unbound antibody is then washed from the sample.
7.2. Duration 5 min þ overnight incubation (day 1) 20 min (day 2) 3.1 Dilute primary antibody in Incubation Buffer and add 0.75–1 ml solution to the tube containing the blocked tissue section. 3.2 Rotate slowly at 4 C, overnight. 3.3 Wash 4 times in PBS – 5 min each wash.
Immunohistochemistry on Freely Floating Fixed Tissue Sections
213
Figure 13.3 Flowchart of Steps 3 and 4.
7.3. Tip Antibody dilutions are often recommended by manufactures but may still have batchto-batch variation. When using antibodies for the first time, test several dilutions to optimize conditions. See Fig. 13.3 for the flowchart of Steps 3 and 4.
8. STEP 4 SECONDARY ANTIBODY LABELING 8.1. Overview Incubation with secondary antibodies amplifies the signal and most often serves as the visualization signal.
8.2. Duration 2.5 h 4.1 Dilute secondary antibody in Incubation Buffer (see product information sheet for dilution). Add 0.75–1 ml of solution to a 1.5-ml round bottom microcentrifuge tube. 4.2 Transfer the sections to the tube and rotate at room temperature for 2 h. If the secondary antibody is conjugated to a fluorophore, all further incubations and washes must be done in the dark to minimize light exposure and photobleaching of the fluorophore. Use aluminum foil to cover samples. 4.3 Wash 4 times in PBS – 5 min each wash.
214
Julia Bachman
9. STEP 5 MOUNT LABELED TISSUE SECTION ON MICROSCOPE SLIDE 9.1. Overview Tissue is aligned and stabilized on microscope slides in order for imaging. Mounting media serves to preserve the tissue and antibody label.
9.2. Duration 10–20 min plus drying time 5.1 Fill a dish with 0.5 PBS and transfer the sections to dish. 5.2 Tilt the slide halfway into the dish and use a paintbrush to gently move section in place on the slide. 5.3 Let the slides dry overnight at room temperature in the dark. 5.4 On the following day, add a drop of mounting media, just enough to cover the tissue once the coverslip is applied to the surface. 5.5 Gently place the coverslip on top of the tissue. Start with one edge against the slide and lower the coverslip gently and slowly, so as not to introduce air bubbles.
Figure 13.4 Flowchart of Step 5.
Immunohistochemistry on Freely Floating Fixed Tissue Sections
215
5.6 Apply a thin layer of nail polish around the perimeter of the coverslip to seal it. Let dry at room temperature in the dark. 5.7 Examine the slide under a fluorescent microscope using the appropriate filters. Photograph representative fields, if possible.
9.3. Tip Excess mounting solution will extend the drying time, and may prevent proper formation of a seal. Note the viscosity of the media, as it will influence the amount needed to spread over the entire tissue area once the coverslip is applied.
9.4. Tip Use fine forceps or syringe needles to handle the coverslip and lower it onto the tissue section. See Fig. 13.4 for the flowchart of Step 5.
Immunohistochemistry on Freely Floating Fixed Tissue Sections Julia Bachman1 Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Theory 2. Equipment 3. Materials 3.1 Solutions & buffers 4. Protocol 4.1 Preparation 4.2 Duration 5. Step 1 Pretreatment of Tissue Section 5.1 Overview 5.2 Duration 6. Step 2 Blocking Nonspecific Binding 6.1 Overview 6.2 Duration 7. Step 3 Primary Antibody Labeling 7.1 Overview 7.2 Duration 7.3 Tip 8. Step 4 Secondary Antibody Labeling 8.1 Overview 8.2 Duration 9. Step 5 Mount Labeled Tissue Section on Microscope Slide 9.1 Overview 9.2 Duration 9.3 Tip 9.4 Tip
208 208 208 209 210 210 210 210 210 210 211 211 211 212 212 212 213 213 213 213 214 214 214 215 215
Abstract Immunohistochemistry on free floating tissue sections is done for many reasons, all of which involve labeling tissue to visualize a certain cell type, protein, or structural component. Visualization is aided by mounting sections on microscope slides for stabilization, and is in most cases necessary for the appropriate use of objectives with a high numerical aperture and high degree of magnification. Methods in Enzymology, Volume 533 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-420067-8.00013-1
#
2013 Elsevier Inc. All rights reserved.
207
208
Julia Bachman
1. THEORY In order to visualize a given component of a tissue sample, the tissue is fixed, sectioned, and labeled. Fixation preserves the molecular organization of the tissue by cross-linking proteins. The most common method of fixation uses 4% paraformaldehyde. Fixation can be done before or after sectioning, depending on the experimental design. Sectioning tissue allows for a more detailed analysis, since thinner sections are more easily penetrated by labeling agents (antibodies and dyes) and by light. Detergent is then used to permeabilize the tissue, which enhances label penetration. The following protocol is for immunolabeling tissue sections – using antibody-based identification to probe particular molecules. The sections are blocked with high amounts of serum to reduce nonspecific binding of subsequent antibody treatments. Primary antibodies specifically label a protein of interest (POI), while secondary antibodies are used for signal amplification and are often conjugated to a visualization signal, such as a fluorophore.
2. EQUIPMENT Flame-polished Pasteur pipette (in hook-shape, for transferring sections) Note: Plastic Pasteur pipettes can also be used to transfer delicate or small tissue (just make sure that the opening is wider than the specimen size and work slowly, so as not to introduce air bubbles into the tissue). Rotator Microscope slides and coverslips (adjust both to size of tissue (length and width) and working distance of microscope (thickness)) Fine paintbrush Glass or hard plastic dish or container with inserts with mesh bottoms (for washing samples, inserts allow for easy transfer of sections between washes) Note: If specimens are small enough (e.g., Drosophila salivary glands), one can do all incubations and washes in a microcentrifuge tube. Change solutions by either pipetting or vacuum aspiration. Aluminum foil
3. MATERIALS Primary antibody (to protein of interest) Appropriate secondary antibody conjugated to a fluorophore Sodium chloride (NaCl)
209
Immunohistochemistry on Freely Floating Fixed Tissue Sections
Potassium chloride (KCl) Sodium phosphate dibasic (Na2HPO4) Potassium phosphate monobasic (KH2PO4) Triton X-100 Normal Goat Serum (NGS. Alternatives: Normal Donkey Serum, Bovine Serum Albumin) Tissue section mounting media (large variety, all commercially available) Nail polish
3.1. Solutions & buffers 10 PBS Component
Final Concentration
Amount
NaCl
136.9 mM
100 g
KCl
2.68 mM
2.5 g
Na2HPO4
10.1 mM
18 g
KH2PO4
1.76 mM
3g
Dissolve the components in 800 ml purified water. Adjust the pH to 7.5 with NaOH. Add purified water to 1 l
PBST Component
Final Concentration
Stock
Amount
PBS
1
10
50 ml
Triton-X-100
0.3%
10%
15 ml
Add to 435 ml purified water
Blocking Buffer Component
Final Concentration
Stock
Amount
NGS
10%
100%
1 ml
PBST
1
1
9 ml
Component
Final Concentration
Stock
Amount
NGS
2%
100%
200 ml
PBST
1
1
9.8 ml
Incubation Buffer
210
Julia Bachman
4. PROTOCOL 4.1. Preparation Tissue fixation varies depending on the desired component to be visualized, tissue type, and tissue thickness. For example, rodents are typically perfused first with saline, followed by fixative (4% paraformaldehyde). The region or organ of interest is dissected out and incubated in cold fixative for an additional 20 min to 2 h. The tissue is then incubated in increasing amounts of sucrose (10%, 20%, and 30%) for several hours or overnight each. Sucrose serves as an isotonic solution that will prevent tissue shrinkage. The tissue is then sectioned using a microtome or vibratome. Fixation after sectioning requires only a brief (10–20 min) incubation in fixative. Use the flame-polished, hooked Pasteur pipette to transfer sections from wash containers to tubes already containing the appropriate solution. See Video 1. http://dx.doi.org/10.1016/B978-0-12-420067-8.00013-1. Make sure to rinse the pipette if transferring sections to multiple tubes containing different antibodies. When transferring sections from tubes back into wash containers, dump contents into container and use a plastic pipette to rinse the tube, making sure all sections have been removed.
4.2. Duration Preparation
Variable
Protocol
2 h (day 1) 3.5 h (day 2)
See Fig. 13.1 for the flowchart of the complete protocol.
5. STEP 1 PRETREATMENT OF TISSUE SECTION 5.1. Overview Tissue is permeabilized by a light treatment of detergent. This allows for better penetration of antibodies and buffers into the tissue.
5.2. Duration 35 min 1.1 Quickly rinse tissue sections in PBST.
Immunohistochemistry on Freely Floating Fixed Tissue Sections
211
Figure 13.1 Flowchart of the complete protocol, including preparation.
1.2 Incubate in fresh PBST for 10–15 min. 1.3 Wash 4 times in PBS – 5 min each wash. See Fig. 13.2 for the flowchart of Steps 1 and 2.
6. STEP 2 BLOCKING NONSPECIFIC BINDING 6.1. Overview Incubating the sample with a high concentration of serum decreases non specific binding of the antibody to the tissue. Other antibodies and components of the serum will bind to ‘stickier’ areas of the tissue thus blocking binding of the antibody used for the specific stain in question.
6.2. Duration About 1 h 2.1 Fill 1.5-ml round bottom microcentrifuge tubes with 1 ml of Blocking Buffer. 2.2 Transfer the tissue sections into tubes.
212
Julia Bachman
Figure 13.2 Flowchart of Steps 1 and 2.
2.3 Rotate at room temperature for 1 h. In the meantime, prepare solutions for incubation with primary antibody. 2.4 Quickly rinse sections in PBS.
7. STEP 3 PRIMARY ANTIBODY LABELING 7.1. Overview Tissue is incubated with antibody raised against specific proteins of interest. The serum concentration is lowered to aid specific binding of the antibody. Any unbound antibody is then washed from the sample.
7.2. Duration 5 min þ overnight incubation (day 1) 20 min (day 2) 3.1 Dilute primary antibody in Incubation Buffer and add 0.75–1 ml solution to the tube containing the blocked tissue section. 3.2 Rotate slowly at 4 C, overnight. 3.3 Wash 4 times in PBS – 5 min each wash.
Immunohistochemistry on Freely Floating Fixed Tissue Sections
213
Figure 13.3 Flowchart of Steps 3 and 4.
7.3. Tip Antibody dilutions are often recommended by manufactures but may still have batchto-batch variation. When using antibodies for the first time, test several dilutions to optimize conditions. See Fig. 13.3 for the flowchart of Steps 3 and 4.
8. STEP 4 SECONDARY ANTIBODY LABELING 8.1. Overview Incubation with secondary antibodies amplifies the signal and most often serves as the visualization signal.
8.2. Duration 2.5 h 4.1 Dilute secondary antibody in Incubation Buffer (see product information sheet for dilution). Add 0.75–1 ml of solution to a 1.5-ml round bottom microcentrifuge tube. 4.2 Transfer the sections to the tube and rotate at room temperature for 2 h. If the secondary antibody is conjugated to a fluorophore, all further incubations and washes must be done in the dark to minimize light exposure and photobleaching of the fluorophore. Use aluminum foil to cover samples. 4.3 Wash 4 times in PBS – 5 min each wash.
214
Julia Bachman
9. STEP 5 MOUNT LABELED TISSUE SECTION ON MICROSCOPE SLIDE 9.1. Overview Tissue is aligned and stabilized on microscope slides in order for imaging. Mounting media serves to preserve the tissue and antibody label.
9.2. Duration 10–20 min plus drying time 5.1 Fill a dish with 0.5 PBS and transfer the sections to dish. 5.2 Tilt the slide halfway into the dish and use a paintbrush to gently move section in place on the slide. 5.3 Let the slides dry overnight at room temperature in the dark. 5.4 On the following day, add a drop of mounting media, just enough to cover the tissue once the coverslip is applied to the surface. 5.5 Gently place the coverslip on top of the tissue. Start with one edge against the slide and lower the coverslip gently and slowly, so as not to introduce air bubbles.
Figure 13.4 Flowchart of Step 5.
Immunohistochemistry on Freely Floating Fixed Tissue Sections
215
5.6 Apply a thin layer of nail polish around the perimeter of the coverslip to seal it. Let dry at room temperature in the dark. 5.7 Examine the slide under a fluorescent microscope using the appropriate filters. Photograph representative fields, if possible.
9.3. Tip Excess mounting solution will extend the drying time, and may prevent proper formation of a seal. Note the viscosity of the media, as it will influence the amount needed to spread over the entire tissue area once the coverslip is applied.
9.4. Tip Use fine forceps or syringe needles to handle the coverslip and lower it onto the tissue section. See Fig. 13.4 for the flowchart of Step 5.
