5. Drain off excess 1X PBS (gently blot PBS off underside of coverslip after draining) and transfer slides/coverslips to humid chamber.

Transcription

1 IMMUNOFLUORESCENCE: SINGLE AND DUAL LABELING WITH MONOCLONAL PRIMARY ANTIBODIES (INDIRECT METHOD) See Developmental Dynamics 206:24-38, Fix sections/coverslips in 3% paraformaldehyde as directed in fixation protocol. Cryoprotected sections (5-10 µm) mounted on gelatin-covered slides should air dry for several minutes before continuing on. Circle each section on slide with PAP pen (not necessary for coverslips). Then rehydrate sections in 1X PBS for 10 min (2 changes). Coverslips containing cell monolayers (e.g. cultured cells) should be washed with 1X PBS (3 times for 5 min) do not need to be rehydrated after fixation. 2. For aldehyde fixed samples, incubate in 50 mm ammonium chloride (in 1X PBS kept at 4 o C) for 30 min at room temp. Use Coplin jar for slides and 6 well plate for coverslips (each well needs 4 ml). 3. Rinse slides in 1X PBS - 3 times for 5 min each. 4. Prepare humid chamber. Line staining tray (or any other sealable plastic box) with gauze, saturate gauze with dh 2 O and then place cottonless swabs on top of gauze. Use 5 swabs/slide or 3 swabs/coverslip. Make up fresh for each experiment. 5. Drain off excess 1X PBS (gently blot PBS off underside of coverslip after draining) and transfer slides/coverslips to humid chamber. 6. Block samples with 20% normal goat serum (NGS) in 1X PBS with 0.5% Triton X-100 (TX- 100). Place about 100 µl of serum-pbs-tx-100 on each section or 200 µl on each coverslip. Be sure to filter the 20% serum through a 0.22 µm syringe filter first. Block sections for 30 min at room temp. in humid chamber. 7. Gently aspirate off serum (we use water aspirator). DO NOT wash in 1X PBS after this step. DO NOT allow sections to dry out at any point. 8. Apply first primary antibody. Make the proper dilutions in 1X PBS with 2% NGS and 0.5% TX-100 (you will need approximately ml of diluted Ab per coverslip or slide). Always centrifuge Ab for this and all subsequent Abs for 1 min to remove any protein complexes or bacteria. Either incubate for 1.5 hrs at room temp. or overnight at 4 o C (1.5 hrs at RT is better because there is less background). Make sure this is done in a humid chamber. 9. Wash slides in 1X PBS+0.5% TX x 5 min washes in Coplin jars or 6 well plates. 11. Drain off excess liquid and place slides in humid chamber. Block with 2-20% NGS in 1X PBS with 0.5% TX-100 (usually use 20% NGS unless I m having trouble with obtaining a positive stain). Block for 30 min at room temperature. 12. Gently aspirate off serum.

2 13. Apply secondary Ab (we use Texas Red conjugated to goat anti-mouse IgG first - Molecular Probes). Dilute secondary Abs with 2% NGS in 1X PBS with 0.5% TX-100 (with cryosections you must use 20% NGS). Incubate for 1 hr at room temp. in humid chamber. NOTE: If only staiing with one round of mab, skip to step Wash slides in 1X PBS+0.5% TX x 5 min in Coplin jars or 6 well plates. If using cryosections, add a final rinse with 1X PBS alone. 15. Drain off excess liquid and place slides in humid chamber. Add 1:20 dilution of goat antimouse IgG Ab (Sigma) to each sample (dilute in 1X PBS + 0.5% TX % NGS). Place humid chamber in cold room overnight. Make note of any samples which have dried out before going on to next step. Dilute goat anti-moue IgG Ab in 1 ml 1X PBS; store in 50 ml one use aliquots in -80 o C. 16. Wash slides in 1X PBS + 0.5% TX x 5 min washes in Coplin jars or 6 well plates. If using cryosections, add a final rinse with 1X PBS alone. 17. Drain off excess liquid and place slides in humid chamber. Add 1:20 dilution of goat antimouse Fab (Jackson ImmunoResearch) fragments. Dilute in 1X PBS + 0.5% TX % NGS. Incubate at room temperature for 1.5 hrs. The FAB fragment comes in liquid form - it should be aliquoted in 1 use aliquots and stored in the -80 o C freezer. 18. Wash slides in 1X PBS + 0.5% TX x 5 min washes in Coplin jars or 6 well plates. If using cryosections, add a final rinse with 1X PBS alone. 19. Block samples with 20% normal goat serum (NGS) in 1X PBS with 0.5% Triton X-100 (TX- 100). Place about 100 µl of serum-1x PBS-TX-100 on each section or 200 µl on each coverslip. Be sure to filter the 20% serum through a 0.22 µm filter first. Block sections for 30 min at room temp. in humid chamber. 19. Drain off excess liquid and place slides in humid chamber. Add the second primary Ab (we had better success using TI-1 followed by TI-4; JLT-12 TnT followed by TI-4; TI-4 followed by Tm (CH1); TI-4 followed by actin) Dilute the second primary Ab in 1X PBS + 0.5% TX % NGS incubate for 1.5 hrs at room temp. (poor results with overnight incubation with second primary Ab). 20. Wash slides in 1X PBS+0.5% TX x 5 min washes in Coplin jars or 6 well plates. 21. Drain off excess liquid and place slides in humid chamber. Block with 20% NGS in 1X PBS with 0.5% TX-100. Block for 30 min at room temperature. 22. Gently aspirate off serum. 23. Apply second secondary Ab (we use FITC conjugated to goat anti-mouse IgG - Molecular Probes). Dilute secondary Abs with 2% NGS in 1X PBS with 0.5% TX-100. Incubate for 1

3 hr at room temp. in humid chamber. 24. Wash 4 x 5 min in 1X PBS alone. 25. Drain off excess liquid. Wipe away liquid from underside of coverslips or a film develops. For slides, add mounting media directly to slide and then cover with rectangular coverslip. For coverslips, add drop of mounting media to a slide and then invert coverslip onto slide. Drain off excess mounting media. Seal edges with clear nailpolish (Wet n Wild). Mounting media is 3 ml glycerol, 1 ml 1X PBS, g p-phenyldiamine. Label and store at -80 o C. 26. Examine using Leitz Aristoplan microscope/biorad confocal microscope. 1X PBS 1 liter: g sodium monophosphate g sodium diphosphate 8.76 g sodium chloride g potassium chloride ph will be , filter (0.45 µm) and store at 4 o C no more than 1 week. Mounting media (make fresh) 1 ml 1X PBS 3 ml glycerol g p-phenyldiamine

4 SUGGESTED ANTIBODY DILUTIONS Secondary Abs: Goat anti-mouse IgG Ab conjugated to TR: 1:100 dilution; Molecular Probes #T862 Goat anti-mouse IgG Ab conjugated to FITC: 1:200 dilution; Sigma #F2012 Goat anti-rabbit IgG Ab conjugated to TR: 1:100 dilution; Molecular Probes #T2767 Goat anti-rabbit IgG Ab conjugated to FITC: 1:200 dilution; Sigma #F0511 Primary Abs: Troponin I Abs: TI-1 (ctni): 1:1000 ; gift of S. Schiaffino TI-4 (generic TnI): 1:500; gift of S. Schiaffino Chemicon Mab 1691 (TnI):1:500-1:1000 Ladenson Abs (TnI) (gift of Ladenson Lab at Washington Univ., St. Louis, MO): 3350 ctni Ab = 1: ctni Ab = 1:2000-1:5000 Tropomyosin: CH1 (monoclonal, sarcomeric; Sigma T9283 from Lin) - appears to crossreact slightly with TnT on Western blots. Dilution for immunofluorescence- 1:200 Actin: monoclonal HUC1-1 (monoclonal from Lessard) - 1:100 polyclonal (Sigma; A2668; recognizes all actins) - 1:500 monoclonal (Sigma, A2172, clone 5C5) - poor results Troponin T: JLT-12 (Sigma; TnT): 1:200 Mab 1695 (Chemicon; TnT): Unknown Myosin Light Chain - polyclonal from K. Chien lab at UCSD - 1:300-1:400 Connexin 43: (Zymed) - 1:500 Myosin: L. Leinwand monoclonal - not used anti-slow myosin Ab (Amersham RPN1168) - recognizes slow SKM, cardiac and smooth muscle myosins - dilution: unknown anti-fast myosin Ab (Amersham RPN 1167) - dilution unknown anti-sarcomeric Ab (polyclonal, Sigma M7648) - poor results with immunoblots, FLAG Ab: 1:500 - supplied by?. Miscellaneous: Goat Serum: Sigma Goat anti-mouse IgG: Sigma M8645 Goat anti-mouse IgG Fab Fragments: Jackson ImmunoResearch

5 FIGURE 1B: DUAL MONOCLONAL LABELING USING TI-4 AND TI-1 ANTI-TnI ANTIBODIES (FLOW CHART) Fix cells in 3% paraformaldehyde - 30 min Wash 3 x 5 min with 1X PBS Treat cells with 50 mm NH 4 Cl - 30 min Wash 3 x 5 min with 1X PBS Block nonspecific binding with 20% normal goat serum (NGS) - 30 min - (diluent=1x PBS=0.5% TX-100) Incubate in TI-1 anti-tni Ab hrs at room temperature * (diluent = 1X PBS + 0.5% TX % NGS) Wash 3 x 5 min with 1X PBS+0.5% TX-100 (Note: if using cryosections add an extra rinse with 1X PBS alone for this step and all subsequent wash steps with 1X PBS+0.5% TX-100) Block nonspecific binding with 2-20% NGS (diluent = 1X PBS+0.5% TX-100) NOTE: If using cryosections, block with 20% NGS Incubate in goat anti-mouse TR-conjugated secondary Ab 1 hr at room temperature (diluent = 1X PBS +0.5% TX % NGS) Note: Use 20% NGS if using cryosections Wash 3 x 5 min with 1X PBS+0.5% TX-100 Wash 3 x 5 min with 1X PBS+0.5% TX-100 End of Day 1 Quench binding sites on primary Ab with goat anti-mouse IgG Ab - overnight incubation at 4 o C (diluent = 1X PBS + 0.5% TX % NGS)

6 Day 2: Wash 3 x 5 min with 1X PBS+ 0.5% TX-100 Quench remaining sites on primary Ab with goat anti-mouse Fab hrs at room temperature (diluent = 1X PBS + 0.5% TX % NGS) Wash 3 x 5 min with 1X PBS + 0.5% TX-100 Block nonspecific binding with 20% NGS (diluent = 1X PBS + 0.5% TX-100) Incubate in TI-4 anti-tni Ab hrs at room temperature Diluent = 1X PBS + 0.5% TX % NGS Wash 3 x 5 min with 1X PBS + 0.5% TX-100 Block nonspecific binding with 20% NGS (diluent = 1X PBS + 0.5% TX-100) Incubate in goat anti-mouse FITC-conjugated Ab 1hr at room temperature (diluent = 1X PBS + 0.5% TX % NGS) Wash 3 x 5 min with 1X PBS alone Mount coverslips, seal with clear nailpolish and examine with Leitz Aristoplan microscope

7 IMMUNOFLUORESCENCE: DUAL LABELING WITH A MONOCLONAL AND POLYCLONAL PRIMARY ANTIBODY (INDIRECT METHOD) 1. Fix sections/coverslips in 3% paraformaldehyde as directed in fixation protocol. Cryoprotected sections (5-10 µm) mounted on gelatin-covered slides should air dry for several minutes before continuing on. Circle each section on slide with PAP pen (not necessary for coverslips). Then rehydrate sections in 1X PBS for 10 min (2 changes). Coverslips containing cell monolayers (e.g. cultured cells) should be washed with 1X PBS (3 times for 5 min) do not need to be rehydrated after fixation. 2. For aldehyde fixed samples, incubate in 50 mm ammonium chloride (in 1X PBS) for 30 min at room temp. Use Coplin jar for slides and 6 well plate for coverslips (each well needs 4 ml). 3. Rinse slides in 1X PBS - 3 times for 5 min each. 4. Prepare humid chamber. Line staining tray (or any other sealable plastic box) with gauze, saturate gauze with dh 2 O and then place cottonless swabs on top of gauze. Use 5 swabs/slide (2 on each end of slide plus one in middle) or 3 swabs/coverslip. Make up fresh for each experiment. 5. Drain off excess 1X PBS (gently blot 1X PBS off underside of coverslip after draining) and transfer slides/coverslips to humid chamber. 6. Block samples with 20% normal goat serum (NGS) in 1X PBS with 0.5% Triton X-100 (TX- 100). Place about 100 µl of serum on each section or 200 µl on each coverslip. Be sure to filter the 20% serum through a 0.22 µm syringe filter first. Block sections for 30 min at room temp. in humid chamber. 7. Gently aspirate off serum (we use water aspirator). DO NOT wash in 1X PBS after this step. DO NOT allow sections to dry out at any point. 8. Apply first primary antibody. Make the proper dilutions in 1X PBS with 2% NGS and 0.5% TX-100. Always centrifuge Ab for 1 min to remove any protein complexes or bacteria. Either incubate for 1.5 hrs at room temp. or overnight at 4 o C (1.5 hrs better). Make sure this is done in a humid chamber. 9. Drain off excess primary Ab #1. Then add the second primary Ab (we had better success using the monoclonal Ab first followed by the polyclonal Ab - less background). Dilute the second primary Ab as described in 8 and incubate for 1.5 hrs at room temp. (poor results with overnight incubation with second primary Ab). 10. Wash slides in 1X PBS+0.5% TX x 5 min washes in Coplin jars or 6 well plates. 11. Drain off excess liquid and place slides in humid chamber. Block with 2% NGS in 1X PBS

8 with 0.5% TX-100. Block for 30 min at room temperature. 12. Gently aspirate off serum. 13. Apply both secondary Abs (we use Texas Red conjugated to goat anti-mouse IgG and FITC conjugated to goat anti-rabbit IgG - Molecular Probes). Dilute secondary Abs with 2% NGS in 1X PBS with 0.5% TX-100. Incubate for 1 hr at room temp. in humid chamber. 14. Wash 4 x 5 min in 1X PBS alone. 15. Drain off excess liquid. Wipe away liquid from underside of coverslips or a film develops. For slides, add mounting media directly to slide and then cover with rectangular coverslip. For coverslips, add drop of mounting media to a slide and then invert coverslip onto slide. Drain off excess mounting media. Seal edges with clear nailpolish (Wet n Wild). Mounting media is 3 ml glycerol, 1 ml 1X PBS, g p-phenyldiamine. Label and store at -80 o C. 16. Examine using Leitz Aristoplan microscope.

9 FIGURE 1A. DUAL LABELLING WITH MONOCLONAL AND POLYCLONAL ANTIBODIES (FLOW CHART) Can be done in 1 day Fix cells in 3% paraformaldehyde - 30 min Wash 3x5 min with 1X PBS Treat cells with 50 mm NH 4 Cl - 30 min Wash 3x5 min with 1X PBS Block nonspecific binding with 20% normal goat serum (NGS) - 30 min (diluent=1x PBS+0.5%TX-100) Incubate with TI-1 or TI-4 anti-tni Ab 1.5 hrs at room temperature (diluent=1x PBS+0.5%TX-100+2%NGS) Drain well to remove excess Ab Incubate with polyclonal anti-myosin light chain Ab 1.5 hrs at room temperature (Same diluent as above) Wash 3x5 min with 1X PBS+0.5% TX-100 Block nonspecific binding with 2% NGS - 30 min (diluent=1x PBS+0.5% TX-100) Incubate with conjugated secondary Abs for 1 hr at room temp. (diluent=1x PBS+0.5%TX %NGS) Secondary Abs: Goat anti-mouse conjugated to Texas Red (TR) and Goat anti-rabbit conjugated to fluorescein isothiocyanate (FITC) Wash 4x5min with 1X PBS Mount coverslips, seal with clear nailpolish and examine on Leitz Aristoplan microscope